Restimulation of cryopreserved tumor infiltrating lymphocytes

ABSTRACT

The present disclosure provides methods for re-stimulating TIL populations that lead to improved phenotype and increased metabolic health of the TILs and provides methods of assaying for TIL populations to determine suitability for more efficacious infusion after re-stimulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/480,941, filed on Sep. 21, 2021, now U.S. Pat. No. 11,266,694, whichis a continuation of U.S. patent application Ser. No. 17/459,988, filedAug. 27, 2021, now U.S. Pat. No. 11,304,980, which is a continuation ofU.S. patent application Ser. No. 17/233,290, filed Apr. 16, 2021, nowU.S. Pat. No. 11,179,419, which is a continuation of U.S. patentapplication Ser. No. 15/751,440, filed Feb. 8, 2018, now U.S. Pat. No.11,026,974, which is a U.S. National Stage entry of International PatentApplication No. PCT/US2017/058610, filed Oct. 26, 2017, which claimspriority to U.S. Provisional Patent Application Nos. 62/413,283 and62/413,387, each filed Oct. 26, 2016, and each entitled “Expansion ofTumor-Infiltrating Lymphocytes and Methods of Using the Same,” and U.S.Provisional Patent Application No. 62/415,452, filed Oct. 31, 2016,entitled “RESTIMULATION OF CRYOPRESERVED TUMOR INFILTRATINGLYMPHOCYTES,” which are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 9, 2021,titled 116983-5004-US15_Sequence_Listing.txt and is 14,171 kilobytes insize.

BACKGROUND OF THE INVENTION

Treatment of bulky, refractory cancers using adoptive transfer of tumorinfiltrating lymphocytes (TILs) represents a powerful approach totherapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev.Immunol. 2006, 6, 383-393. A large number of TILs are required forsuccessful immunotherapy, and a robust and reliable process is neededfor commercialization. This has been a challenge to achieve because oftechnical, logistical, and regulatory issues with cell expansion.IL-2-based TIL expansion followed by a “rapid expansion process” (REP)has become a preferred method for TIL expansion because of its speed andefficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al.,J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008,26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al.,J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-foldexpansion of TILs over a 14-day period, although it requires a largeexcess (e.g., 200-fold) of irradiated allogeneic peripheral bloodmononuclear cells (PBMCs), often from multiple donors, as feeder cells,as well as anti-CD3 antibody (OKT3) and high doses of IL-2. Dudley, etal., J. Immunother. 2003, 26, 332-42.

TILs that have undergone an REP procedure have produced successfuladoptive cell therapy following host immunosuppression in patients withmelanoma. Current infusion acceptance parameters rely on readouts of thecomposition of TILs (e.g., CD28, CD8, or CD4 positivity) and on foldexpansion and viability of the REP product.

However, current REP protocols, as well as current TIL expansionprotocols generally, give little insight into the health of the TIL thatwill be infused into the patient. T cells undergo a profound metabolicshift during the course of their maturation from naïve to effector Tcells (see Chang, et al., Nat. Immunol. 2016, 17, 364, hereby expresslyincorporated in its entirety, and in particular for the discussion andmarkers of anaerobic and aerobic metabolism). For example, naïve T cellsrely on mitochondrial respiration to produce ATP, while mature, healthyeffector T cells such as TIL are highly glycolytic, relying on aerobicglycolysis to provide the bioenergetics substrates they require forproliferation, migration, activation, and anti-tumor efficacy.

In addition, these expanded cell populations can be cryopreserved,leading to ease of use, long-term storage for multiple reinfusions intopatients with recurrent disease, and other considerations. However,current infusion acceptance parameters rely on readouts of thecomposition of TILs and on fold-expansion and viability of the expandedTIL based product. These measures give little insight into the health ofthe TIL that will be infused into the patient, and little is known aboutthe effects of cryopreservation on TIL populations.

Accordingly, the present invention is directed to methods for expandingand re-stimulating TIL populations that lead to improved phenotype andincreased metabolic health of the TILs and towards methods of assayingfor TIL populations to determine suitability for more efficaciousinfusion after re-stimulation.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for expanding TILs in larger,sometimes therapeutic, populations in combination with optionalcryopreservation.

According to the present disclosure, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILscomprising the following steps is provided:

-   -   (i) obtaining a first population of TILs from a tumor resected        from a patient;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold or 100-fold greater in number than the        second population of TILs, and wherein the second expansion is        performed for at least 14 days in order to obtain the third        population of TILs, wherein the third population of TILs is a        therapeutic population of TILs which comprises an increased        subpopulation of effector T cells and/or central memory T cells        relative to the second population of TILs.

In some embodiments, the method further comprises:

-   -   (iv) performing an additional second expansion by supplementing        the cell culture medium of the third population of TILs with        additional IL-2, additional OKT-3, and additional APCs, wherein        the additional second expansion is performed for at least 14        days to obtain a larger therapeutic population of TILs than        obtained in step (iii), wherein the larger therapeutic        population of TILs comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        third population of TILs.

In some embodiments, after step (iii), the cells are removed from thecell culture and cryopreserved in a storage medium prior to performingstep (iv).

In some embodiments, the cells are thawed prior to performing step (iv).

In some embodiments, step (iv) is repeated one to four times in order toobtain sufficient TILs in the therapeutic population of TILs for atherapeutically effective dosage of the TILs.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 40 days to about 50 days. In some embodiments,steps (i) through (iii) or (iv) are performed within a period of about42 days to about 48 days. In some embodiments, steps (i) through (iii)or (iv) are performed within a period of about 42 days to about 45 days.In some embodiments, steps (i) through (iii) or (iv) are performedwithin about 44 days.

In some embodiments, the cells from steps (iii) or (iv) express CD4,CD8, and TCR α β at levels similar to freshly harvested cells.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are added tothe cell culture on any of days 9 through 17 in step (iii).

In some embodiments, the effector T cells and/or central memory T cellsin the therapeutic population of TILs in step (iv) exhibit one or morecharacteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T cells and/orcentral memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a high-affinity T cell receptor.

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a chimeric antigen receptor (CAR)comprising a single chain variable fragment antibody fused with at leastone endodomain of a T-cell signaling molecule.

In some embodiments, the therapeutic population of TILs are infused intoa patient.

In some embodiments, step (iii) further comprises a step of removing thecells from the cell culture medium.

In some embodiments, step (iii) is repeated one to four times in orderto obtain sufficient TILs in the therapeutic population of TILs for atherapeutically effective dosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

The present disclosure also provides a population of expanded TILs madeaccording to the method of claim 1.

The present disclosure also provides a population of expanded TILs madeaccording to the method of claim 1, wherein the expanded TILs have atleast a two-fold increase in basal glycolysis as compared to thawedcryopreserved TILs.

The present disclosure also provides methods for assessing the metabolicactivity of a TIL cell population made according to the methodsdescribed herein, comprising measuring the basal glycolysis of thecells.

The present disclosure also provides methods for assessing the metabolicactivity of a TIL cell population made according to the methodsdescribed herein, comprising measuring the basal respiration of thecells.

The present disclosure also provides methods for assessing the metabolicactivity of a TIL cell population made according to the methodsdescribed herein, comprising measuring the spare respiratory capacity(SRC) of the cells.

The present disclosure also provides methods for assessing the metabolicactivity of a TIL cell population made according to the methodsdescribed herein, comprising measuring the glycolytic reserve of thecells.

The present disclosure also provides a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILscomprising:

-   -   (i) performing a first expansion by culturing a first population        of TILs from a tumor resected from a patient in a cell culture        medium comprising IL-2 to obtain a second population of TILs;        and    -   (ii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs) to obtain a        third population of TILs, wherein the third population of TILs        is at least 50-fold or 100-fold greater in number than the        second population of TILs, and wherein the second expansion is        performed for at least 14 days in order to obtain the third        population of TILs, wherein the third population of TILs is a        therapeutic population of TILs which comprises an increased        subpopulation of effector T cells and/or central memory T cells        relative to the second population of TILs.

In some embodiments, the method further comprises:

-   -   (iii) performing an additional second expansion of the third        population of TILs by supplementing the cell culture medium of        the third population of TILs with additional IL-2, additional        OKT-3, and additional APCs, wherein the additional second        expansion is performed for at least 14 days to obtain a larger        therapeutic population of TILs than obtained in step (ii),        wherein the larger therapeutic population of TILs exhibits an        increased subpopulation of effector T cells and/or central        memory T cells relative to the third population of TILs.

In some embodiments, the cells from the cell culture medium in step (ii)are removed and cryopreserved in a storage medium prior to step (iii).

In some embodiments, the cells are thawed prior to step (iii).

In some embodiments, step (ii) is repeated one to four times in order toobtain sufficient TILs in the therapeutic population of TILs for atherapeutically effective dosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, the APCs are peripheral blood mononuclear cells(PBMCs).

In some embodiments, the effector T cells and/or central memory T cellsexhibit one or more characteristics selected from the group consistingof expression of CD27, expression of CD28, longer telomeres, increasedCD57 expression, and decreased CD56 expression, relative to effector Tcells and/or central memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

The present disclosure also provides a method for treating a subjectwith cancer comprising administering expanded tumor infiltratinglymphocytes (TILs) comprising:

-   -   (i) obtaining a first population of TILs from a tumor resected        from a patient;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs;    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold or 100-fold greater in number than the        second population of TILs, and wherein the second expansion is        performed for at least 14 days in order to obtain the third        population of TILs, wherein the third population of TILs is a        therapeutic population of TILs which comprises an increased        subpopulation of effector T cells and/or central memory T cells        relative to the second population of TILs; and    -   (iv) administering a therapeutically effective dosage of the        third population of TILs to the patient.

In some embodiments, the method further comprises prior to step (iv) astep of performing an additional second expansion by supplementing thecell culture medium of the third population of TILs with additionalIL-2, additional OKT-3, and additional APCs, wherein the additionalsecond expansion is performed for at least 14 days to obtain a largertherapeutic population of TILs than obtained in step (iii), wherein thelarger therapeutic population of TILs comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the third population of TILs.

In some embodiments, after step (ii) the cells are removed from the cellculture medium and cryopreserved in a storage medium prior to theadditional second expansion according to the methods described herein.

In some embodiments, the cells are thawed prior to the additional secondexpansion of according to the methods described herein.

In some embodiments, step (iii) is repeated one to four times in orderto obtain sufficient TILs in the therapeutic population of TILs for atherapeutically effective dosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, the APCs are peripheral blood mononuclear cells(PBMCs).

In some embodiments, the effector T cells and/or central memory T cellsexhibit one or more characteristics selected from the group consistingof expression of CD27, expression of CD28, longer telomeres, increasedCD57 expression, and decreased CD56 expression, relative to effector Tcells and/or central memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the cancer is selected from the group consisting ofmelanoma, cervical cancer, head and neck cancer, glioblastoma, ovariancancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer,triple negative breast cancer, and non-small cell lung carcinoma.

The present disclosure also provides a method for treating a subjectwith cancer comprising administering expanded tumor infiltratinglymphocytes (TILs) comprising:

-   -   (i) performing a first expansion by culturing a first population        of TILs from a tumor resected from a patient in a cell culture        medium comprising IL-2 to obtain a second population of TILs;    -   (ii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs) to obtain a        third population of TILs, wherein the third population of TILs        is at least 50-fold or 100-fold greater in number than the        second population of TILs, and wherein the second expansion is        performed for at least 14 days in order to obtain the third        population of TILs, wherein the third population of TILs is a        therapeutic population of TILs which comprises an increased        subpopulation of effector T cells and/or central memory T cells        relative to the second population of TILs; and    -   (iii) administering a therapeutically effective dosage of the        therapeutic population of TILs to the patient.

In some embodiments, the method further comprises prior to step (iii) astep of performing an additional second expansion by supplementing thecell culture medium of the third population of TILs with additionalIL-2, additional OKT-3, and additional APCs, wherein the additionalsecond expansion is performed for at least 14 days to obtain a largertherapeutic population of TILs than obtained in step (ii), wherein thelarger therapeutic population of TILs comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the third population of TILs.

In some embodiments, the cells from the cell culture medium in step (ii)are removed and cryopreserved in a storage medium prior to theadditional second expansion as described herein.

In some embodiments, the cells are thawed prior to the additional secondexpansion as described herein.

In some embodiments, step (ii) is repeated one to four times in order toobtain sufficient TILs in the therapeutic population of TILs for atherapeutically effective dosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, the APCs are peripheral blood mononuclear cells(PBMCs).

In some embodiments, the effector T cells and/or central memory T cellsexhibit one or more characteristics selected from the group consistingof expression of CD27, expression of CD28, longer telomeres, increasedCD57 expression, and decreased CD56 expression, relative to effector Tcells and/or central memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the cancer is selected from the group consisting ofmelanoma, cervical cancer, head and neck cancer, glioblastoma, ovariancancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer,triple negative breast cancer, and non-small cell lung carcinoma.

The present invention also provides assay methods for determining TILviability. The present disclosure provides methods for assaying TILs forviability by expanding tumor infiltrating lymphocytes (TILs) into alarger population of TILs comprising:

-   -   (i) obtaining a first population of TILs which has been        previously expanded;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold or 100-fold greater in number than the        second population of TILs, and wherein the second expansion is        performed for at least 14 days in order to obtain the third        population of TILs, wherein the third population of TILs        comprises an increased subpopulation of effector T cells and/or        central memory T cells relative to the second population of        TILs, and wherein the third population is further assayed for        viability.

In some embodiments, the method further comprises:

-   -   (iv) performing an additional second expansion by supplementing        the cell culture medium of the third population of TILs with        additional IL-2, additional OKT-3, and additional APCs, wherein        the additional second expansion is performed for at least 14        days to obtain a larger population of TILs than obtained in step        (iii), wherein the larger population of TILs comprises an        increased subpopulation of effector T cells and/or central        memory T cells relative to the third population of TILs, and        wherein the third population is further assayed for viability.

In some embodiments, prior to step (i), the cells are cryopreserved.

In some embodiments, the cells are thawed prior to performing step (i).

In some embodiments, step (iv) is repeated one to four times in order toobtain sufficient TILs for analysis.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 40 days to about 50 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 48 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 45 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin about 44 days.

In some embodiments, the cells from steps (iii) or (iv) express CD4,CD8, and TCR α β at levels similar to freshly harvested cells.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 17 in step (iii).

In some embodiments, the effector T cells and/or central memory T cellsin the larger population of TILs in step (iv) exhibit one or morecharacteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T cells, and/orcentral memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a high-affinity T cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a chimeric antigen receptor (CAR)comprising a single chain variable fragment antibody fused with at leastone endodomain of a T-cell signaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability.

In some embodiments, the TILs are assayed for viability aftercryopreservation.

In some embodiments, the TILs are assayed for viability aftercryopreservation and after step (iv).

According to the present disclosure, a method for assaying TILs forviability and/or further use in administration to a subject. In someembodiments, the method for assay tumor infiltrating lymphocytes (TILs)comprises:

-   -   (i) obtaining a first population of TILs;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs;

(iv) harvesting, washing, and cryopreserving the third population ofTILs;

-   -   (v) storing the cryopreserved TILs at a cryogenic temperature;    -   (vi) thawing the third population of TILs to provide a thawed        third population of TILs; and    -   (vii) performing an additional second expansion of a portion of        the thawed third population of TILs by supplementing the cell        culture medium of the third population with IL-2, OKT-3, and        APCs for a reREP period of at least 3 days, wherein the third        expansion is performed to obtain a fourth population of TILs,        wherein the number of TILs in the fourth population of TILs is        compared to the number of TILs in the third population of TILs        to obtain a ratio;    -   (viii) determining based on the ratio in step (vii) whether the        thawed population of TILs is suitable for administration to a        patient;    -   (ix) administering a therapeutically effective dosage of the        thawed third population of TILs to the patient when the ratio of        the number of TILs in the fourth population of TILs to the        number of TILs in the third population of TILs is determined to        be greater than 5:1 in step (viii).

In some embodiments, the reREP period is performed until the ratio ofthe number of TILs in the fourth population of TILs to the number ofTILs in the third population of TILs is greater than 50:1.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, steps (i) through (vii) are performed within aperiod of about 40 days to about 50 days. In some embodiments, steps (i)through (vii) are performed within a period of about 42 days to about 48days. In some embodiments, steps (i) through (vii) are performed withina period of about 42 days to about 45 days. In some embodiments, steps(i) through (vii) are performed within about 44 days.

In some embodiments, the cells from steps (iii) or (vii) express CD4,CD8, and TCR α β at levels similar to freshly harvested cells. In someembodiments the cells are TILs.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are added tothe cell culture on any of days 9 through 17 in step (iii).

In some embodiments, the effector T cells and/or central memory T cellsin the larger population of TILs in steps (iii) or (vii) exhibit one ormore characteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T cells, and/orcentral memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding ahigh-affinity T cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding achimeric antigen receptor (CAR) comprising a single chain variablefragment antibody fused with at least one endodomain of a T-cellsignaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability after step(vii).

The present disclosure also provides further methods for assaying TILs.In some embodiments, the disclosure provides a method for assaying TILscomprising:

-   -   (i) obtaining a portion of a first population of cryopreserved        TILs;    -   (ii) thawing the portion of the first population of        cryopreserved TILs;    -   (iii) performing a first expansion by culturing the portion of        the first population of TILs in a cell culture medium comprising        IL-2, OKT-3, and antigen presenting cells (APCs) for a reREP        period of at least 3 days, to produce a second population of        TILs, wherein the portion from the first population of TILs is        compared to the second population of TILs to obtain a ratio of        the number of TILs, wherein the ratio of the number of TILs in        the second population of TILs to the number of TILs in the        portion of the first population of TILs is greater than 5:1;    -   (iv) determining based on the ratio in step (iii) whether the        first population of TILs is suitable for use in therapeutic        administration to a patient;    -   (v) determining the first population of TILs is suitable for use        in therapeutic administration when the ratio of the number of        TILs in the second population of TILs to the number of TILs in        the first population of TILs is determined to be greater than        5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the secondpopulation of TILs to the number of TILs in the portion of the firstpopulation of TILs is greater than 50:1.

In some embodiments, the method further comprises performing expansionof the entire first population of cryopreserved TILs from step (i)according to the methods as described in any of the embodiments providedherein.

In some embodiments, the method further comprises administering theentire first population of cryopreserved TILs from step (i) to thepatient.

The present disclosure also provides further methods for assaying TILs.In some embodiments, the disclosure provides a method for assaying TILscomprising:

-   -   (i) obtaining a portion of a first population of cryopreserved        TILs;    -   (ii) thawing the portion of the first population of        cryopreserved TILs;    -   (iii) performing a first expansion by culturing the portion of        the first population of TILs in a cell culture medium comprising        IL-2, OKT-3, and antigen presenting cells (APCs) for a reREP        period of at least 3 days, to produce a second population of        TILs, wherein the portion from the first population of TILs is        compared to the second population of TILs to obtain a ratio of        the number of TILs, wherein the ratio of the number of TILs in        the second population of TILs to the number of TILs in the        portion of the first population of TILs is greater than 5:1;    -   (iv) determining based on the ratio in step (iii) whether the        first population of TILs is suitable for use in therapeutic        administration to a patient; and    -   (v) therapeutically administering the remainder of the first        population of TILs to the patient when the ratio of the number        of TILs in the second population of TILs to the number of TILs        in the first population of TILs is determined to be greater than        5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the secondpopulation of TILs to the number of TILs in the portion of the firstpopulation of TILs is greater than 50:1.

In some embodiments, the method further comprises performing expansionof the entire first population of cryopreserved TILs from step (i)according to the methods of any of the preceding claims.

In some embodiments, the method further comprises administering theentire first population of cryopreserved TILs from step (i) to thepatient.

In some embodiments, the method further comprised the step of assessingthe metabolic health of the second population of TILs.

In some embodiments, the method further comprises the step of assessingthe phenotype of the second population of TILs.

In some embodiments, the antigen presenting cells are allogeneicperipheral blood mononuclear cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the results from Example 1. As the Table shows, followingthe antigen restimulation rapid expansion protocol (“reREP”), the TILsexhibit a marked enhancement in their glycolytic respiration. SRC=sparerespiratory capacity.

FIG. 2: Composition of fresh vs. thawed TIL. TIL were stained for TCRαβand CD56 to define T-cell and NK populations. The data shown areaverages of 6 individual TILs.

FIG. 3: Memory phenotype is defined by CD45RA and CCR7 Expression. CD4and CD8 TIL are mainly Effector Memory (EM) This remains the same in thethawed TIL. Each point is one sample analyzed. No significant differenceis found in a Wilcoxon matched-pairs signed rank test.

FIG. 4: Pearson's correlation of CD4, CD8, CD4+CD28+, and CD8+CD28+frequency between fresh and thawed TIL. Cells were stained with abovemarkers. Each dot represents one individual with the fresh value on thex axis and the thawed value on the y axis. The fit line was drawn usinglinear regression analysis.

FIG. 5: Comparable Activation Markers on Fresh and Thawed TILs. Nosignificant difference in activation status of fresh vs. thawed TIL wasfound using a Wilcoxon Matched-Pairs Rank Test. Each point representsone sample analyzed and is shown as mean+/−SEM.

FIGS. 6A and 6B: Maintenance of LAG-3 Staining FollowingCryopreservation and Thaw. A: LAG-3 staining of CD8 TIL. B: % frequencyof regulatory molecules of the CD4 and CD8 populations on fresh andthawed TIL. CD8+TIM-3+ and CD8+LAG-3+ thawed TIL have a lower % thanfresh TIL. Mann-Whitney statistical test.

FIG. 7: Remarkably stable tumor-infiltrating lymphocytes (TIL) forinfusion phenotype following cryopreservation.

FIG. 8: Scatter plot showing phenotypic characterization of reREP TILs.Q1 shows 19.0% CD45RA⁺/CCR7⁻; Q2 shows 0.066% CD45RA⁺/CCR7⁺; Q4 shows80.6% CD45RA⁻/CCR7⁻; and Q3 shows 0.36% CD45RA⁻/CCR7⁺.

FIG. 9: Diagram and data showing the phenotypic characterization ofreREP TILs, during the first and second expansion phases 0.08%CD45RA⁺/CCR7⁻; 0.03% CD45RA⁺/CCR7⁺; 73.97% CD45RA⁻/CCR7⁻; and 25.91%CD45RA⁻/CCR7⁺ at Day 14, after the first expansion but prior to thesecond expansion. Proliferation of CM or EM TIL in the repeat ReREP.Central Memory (CM) TIL and Effector Memory (EM) TIL were tested for theproliferation capacity using repeat ReREP. Briefly, 1.3×10⁶ Post REP TILwere co-culture with 1.3×10⁷ PBMC feeders (CFSE labelled), OKT3 (30ng/nl) and rhIL-2 (3000 IU/ml), culture was incubated for 14 days. OnDay 14, central memory TIL and effector memory TIL were gated for L/DAqua−/CFSE−/TCRα/β+/CD45RA−/CCR7+ and L/DAqua−/CFSE−/TCRα/β+/CD45RA−/CCR7− population respectively and flowcytometry sorted. Purity of the cell population was 97%. 1×10⁴ flowsorted CM or EM or unsorted TIL were then cultured 1×10⁶ PBMC feeders,OKT3 (30 ng/nl) and IL-2 (3000 IU/ml) in triplicates for 7 days. Cellwere counted and recorded. Central memory TIL were more proliferativewhen compared to Effector memory TIL. We are repeating this experimentwith more post REP TIL lines.

FIGS. 10A and 10B: Phenotypic characterization of TILs during ReREP.Cells were gated on Aqua−/TCRα/β+/CD4+ or CD8+ to show Central MemoryTILs (CD45RA⁻ CCR7⁺) or Effector Memory TILs (CD45RA⁻CCR7⁻) memoryphenotype. Student “t” was used to calculate statistical significance.*p<0.05, ns non-significant.

FIG. 11: Exemplary schematic of the TIL preparation process, sometimesreferred to herein as the 1C process.

FIG. 12: Successful expansion of TILs from non-melanoma tumors. Datashows the distribution of TIL (CD4+/CD8+) in non-melanoma tumors.

FIG. 13: Non-melanoma TILs expressed CD27 and CD38, consistent withyoung TILs.

FIG. 14: Activated TILs skew towards effector memory population.

FIG. 15: Fresh versus reREP TIL phenotypes.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Adoptive cell therapy utilizing TILs cultured ex vivo by the RapidExpansion Protocol (REP) has produced successful adoptive cell therapyfollowing host immunosuppression in patients with melanoma. Currentinfusion acceptance parameters rely on readouts of the composition ofTILs (e.g., CD28, CD8, or CD4 positivity) and on the numerical folds ofexpansion and viability of the REP product.

Current REP protocols give little insight into the health of the TILthat will be infused into the patient. T cells undergo a profoundmetabolic shift during the course of their maturation from naïve toeffector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, herebyexpressly incorporated in its entirety, and in particular for thediscussion and markers of anaerobic and aerobic metabolism). Forexample, naïve T cells rely on mitochondrial respiration to produce ATP,while mature, healthy effector T cells such as TIL are highlyglycolytic, relying on aerobic glycolysis to provide the bioenergeticssubstrates they require for proliferation, migration, activation, andanti-tumor efficacy.

Previous papers report that limiting glycolysis and promotingmitochondrial metabolism in TILs prior to transfer is desirable as cellsthat are relying heavily on glycolysis will suffer nutrient deprivationupon adoptive transfer which results in a majority of the transferredcells dying. Thus, the art teaches that promoting mitochondrialmetabolism might promote in vivo longevity and in fact suggests usinginhibitors of glycolysis before induction of the immune response. SeeChang et al. (Chang, et al., Nat. Immunol. 2016, 17(364), 574-582).

The present invention is directed in preferred aspects to novel methodsof augmenting REPs with an additional restimulation protocol, sometimesreferred to herein as a “restimulation Rapid Expansion Protocol” or“reREP”, which leads surprisingly to expanded memory T cell subsets,including the central memory (CD45RA⁻CCR7⁺) or effector memory(CD45RA⁻CCR7⁻) phenotypes, and/or to marked enhancement in theglycolytic respiration as compared to freshly harvested TILs or thawedcryopreserved TILs for the restimulated TILs (sometimes referred toherein as “reTILs”). That is, by using a reREP procedure (i.e., aprocedure comprising a first expansion and a second expansion) oncryopreserved TILs, patients can receive highly metabolically active,healthy TILs, leading to more favorable outcomes.

The present invention is further directed in some embodiments to methodsfor evaluating and quantifying this increase in metabolic health. Thus,the present invention provides methods of assaying the relative healthof a TIL population using one or more general evaluations of metabolism,including, but not limited to, rates and amounts of glycolysis,oxidative phosphorylation, spare respiratory capacity (SRC) andglycolytic reserve.

Furthermore, the present invention is further directed in someembodiments to methods for evaluating and quantifying this increase inmetabolic health. Thus, the present invention provides methods ofassaying the relative health of a TIL population using one or moregeneral evaluations of metabolism, including, but not limited to, ratesand amounts of glycolysis, oxidative phosphorylation, spare respiratorycapacity (SRC), and glycolytic reserve.

In addition, optional additional evaluations include, but are notlimited to, ATP production, mitochondrial mass and glucose uptake.

In some cases, the reREP cell population with increased metabolic healthare infused into a patient as is generally known in the art.

II. Definitions

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8+ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussedherein.

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient. TILS may further becharacterized by potency—for example, TILS may be considered potent if,for example, interferon (IFN) release is greater than about 50 pg/mL,greater than about 100 pg/mL, greater than about 150 pg/mL, or greaterthan about 200 pg/mL. Interferon can include interferon gamma (IFNγ).

By “cryopreserved TILs” herein is meant that TILs, either primary, bulk,or expanded (REP TILs), are treated and stored in the range of about−150° C. to −60° C. General methods for cryopreservation are alsodescribed elsewhere herein, including in the Examples. For clarity,“cryopreserved TILs” are distinguishable from frozen tissue sampleswhich may be used as a source of primary TILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs thatwas previously cryopreserved and then treated to return to roomtemperature or higher, including but not limited to cell culturetemperatures or temperatures wherein TILs may be administered to apatient.

By “population of cells” (including TILs) herein is meant a number ofcells that share common traits. In general, populations generally rangefrom 1×10⁶ to 1×10¹⁰ in number, with different TIL populationscomprising different numbers. For example, initial growth of primaryTILs in the presence of IL-2 results in a population of bulk TILs ofroughly 1×10⁸ cells. REP expansion is generally done to providepopulations of 1.5×10⁹ to 1.5×10¹⁰ cells for infusion.

In general, TILs are initially obtained from a patient tumor sample(“primary TILs”) and then expanded into a larger population for furthermanipulation as described herein, optionally cryopreserved, restimulatedas outlined herein and optionally evaluated for phenotype and metabolicparameters as an indication of TIL health.

In general, the harvested cell suspension is called a “primary cellpopulation” or a “freshly harvested” cell population.

In general, as discussed herein, the TILs are initially prepared byobtaining a primary population of TILs from a tumor resected from apatient as discussed herein (the “primary cell population” or “firstcell population”). This is followed with an initial bulk expansionutilizing a culturing of the cells with IL-2, forming a secondpopulation of cells (sometimes referred to herein as the “bulk TILpopulation” or “second population”).

The term “cytotoxic lymphocyte” includes cytotoxic T (CTL) cells(including CD8⁺ cytotoxic T lymphocytes and CD4⁺ T-helper lymphocytes),natural killer T (NKT) cells and natural killer (NK) cells. Cytotoxiclymphocytes can include, for example, peripheral blood-derivedα/βTCR-positive or α/βTCR-positive T cells activated by tumor associatedantigens and/or transduced with tumor specific chimeric antigenreceptors or T-cell receptors, and tumor-infiltrating lymphocytes(TILs).

The term “central memory T cell” refers to a subset of T cells that inthe human are CD45RO+ and constitutively express CCR7 (CCR7 hi) andCD62L (CD62 hi). The surface phenotype of central memory T cells alsoincludes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors forcentral memory T cells include BCL-6, BCL-6B, MBD2, and BMII. Centralmemory T cells primarily secret IL-2 and CD40L as effector moleculesafter TCR triggering. Central memory T cells are predominant in the CD4compartment in blood, and in the human are proportionally enriched inlymph nodes and tonsils.

The term “effector memory T cell” refers to a subset of human ormammalian T cells that, like central memory T cells, are CD45R0+, buthave lost the constitutive expression of CCR7 (CCR7lo) and areheterogeneous or low for CD62L expression (CD62Llo). The surfacephenotype of central memory T cells also includes TCR, CD3, CD127(IL-7R), and IL-15R. Transcription factors for central memory T cellsinclude BLIMP1. Effector memory T cells rapidly secret high levels ofinflammatory cytokines following antigenic stimulation, includinginterferon-γ, IL-4, and IL-5. Effector memory T cells are predominant inthe CD8 compartment in blood, and in the human are proportionallyenriched in the lung, liver, and gut. CD8+ effector memory T cells carrylarge amounts of perforin. The term “closed system” refers to a systemthat is closed to the outside environment. Any closed system appropriatefor cell culture methods can be employed with the methods of the presentinvention. Closed systems include, for example, but are not limited toclosed G-containers. Once a tumor segment is added to the closed system,the system is no opened to the outside environment until the TILs areready to be administered to the patient.

The terms “peripheral blood mononuclear cells” and “PBMCs” refers to aperipheral blood cell having a round nucleus, including lymphocytes (Tcells, B cells, NK cells) and monocytes. Preferably, the peripheralblood mononuclear cells are irradiated allogeneic peripheral bloodmononuclear cells.

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are described herein.

In some embodiments, methods of the present disclosure further include a“pre-REP” stage in which tumor tissue or cells from tumor tissue aregrown in standard lab media (including without limitation RPMI) andtreated the with reagents such as irradiated feeder cells and anti-CD3antibodies to achieve a desired effect, such as increase in the numberof TILS and/or an enrichment of the population for cells containingdesired cell surface markers or other structural, biochemical orfunctional features. The pre-REP stage may utilize lab grade reagents(under the assumption that the lab grade reagents get diluted out duringa later REP stage), making it easier to incorporate alternativestrategies for improving TIL production. Therefore, in some embodiments,the disclosed TLR agonist and/or peptide or peptidomimetics can beincluded in the culture medium during the pre-REP stage. The pre-REPculture can in some embodiments, include IL-2.

The present invention is directed in preferred aspects to novel methodsof augmenting REPs with an additional restimulation protocol, sometimesreferred to herein as a “restimulation Rapid Expansion Protocol” or“reREP”, which leads surprisingly to expanded memory T cell subsets,including the memory effector T cell subset, and/or to markedenhancement in the glycolytic respiration as compared to freshlyharvested TILs or thawed cryopreserved TILs for the restimulated TILs(sometimes referred to herein as “reTILs”). That is, by using a reREPprocedure on cryopreserved TILs, patients can receive highlymetabolically active, healthy TILs, leading to more favorable outcomes.Such restimulation protocols, also referred to herein as additional“expansions” of the cell populations, are described in further detailherein.

The terms “fragmenting,” “fragment,” and “fragmented,” as used herein todescribe processes for disrupting a tumor, includes mechanicalfragmentation methods such as crushing, slicing, dividing, andmorcellating tumor tissue as well as any other method for disrupting thephysical structure of tumor tissue. The term “in vivo” refers to anevent that takes place in a subject's body.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “anti-CD3 antibody” refers to an antibody or variant thereof,e.g., a monoclonal antibody and including human, humanized, chimeric ormurine antibodies which are directed against the CD3 receptor in the Tcell antigen receptor of mature T cells. Anti-CD3 antibodies includeOKT-3, also known as muromonab, and UCHT-1. Other anti-CD3 antibodiesinclude, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to amonoclonal antibody or biosimilar or variant thereof, including human,humanized, chimeric, or murine antibodies, directed against the CD3receptor in the T cell antigen receptor of mature T cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof. The amino acid sequences of the heavy and lightchains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).A hybridoma capable of producing OKT-3 is deposited with the AmericanType Culture Collection and assigned the ATCC accession number CRL 8001.A hybridoma capable of producing OKT-3 is also deposited with EuropeanCollection of Authenticated Cell Cultures (ECACC) and assigned CatalogueNo. 86022706.

TABLE 1 Amino acid sequences of muromonab. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ IDQVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY  60NO: 1NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120MuromonabKTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180heavyYTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240chainPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK                                  450SEQ IDQIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH  60NO: 2FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120MuromonabSEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180lightTKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC                              213chain

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2, and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO:3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The amino acid sequence ofaldesleukin suitable for use in the invention is given in Table 2 (SEQID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, asdescribed herein, including the pegylated IL2 prodrug NKTR-214,available from Nektar Therapeutics, South San Francisco, Calif., USA.NKTR-214 and pegylated IL-2 suitable for use in the invention isdescribed in U.S. Patent Application Publication No. US 2014/0328791 A1and International Patent Application Publication No. WO 2012/065086 A1,the disclosures of which are incorporated by reference herein.Alternative forms of conjugated IL-2 suitable for use in the inventionare described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and4,902,502, the disclosures of which are incorporated by referenceherein. Formulations of IL-2 suitable for use in the invention aredescribed in U.S. Pat. No. 6,706,289, the disclosure of which isincorporated by reference herein.

TABLE 2 Amino acid sequences of interleukins. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL  60recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120human IL-2RWITFCQSII STLT                                                   134(rhIL-2) SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE  60AldesleukinELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120ITFSQSIIST LT                                                     132SEQ ID NO:5MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH  60recombinantEKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120human IL-4MREKYSKCSS                                                        130(rhIL-4) SEQ ID NO: 6MDCDIEGKDG KQYESVIMVS IDQLLDSMKE IGSNCLNNEF EFFKRHICDA NKEGMFLFRA  60recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAAIGEAQP TKSLEENKSL 120human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH                             153(rhIL-7) SEQ ID NO: 7MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI  60recombinantHDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINIS      115human IL-15 (rhIL-15) SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG  60recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120human IL-21HLSSRTHGSE DS                                                     132(rhIL-21)

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naïve helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMHC expression, and induces class switching to IgE and IgG1 expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acidsequence of recombinant human IL-4 suitable for use in the invention isgiven in Table 2 (SEQ ID NO:5).

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of IL-7 receptor alpha and common gamma chain receptor, whichin a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human IL-4suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No.Gibco PHC0071). The amino acid sequence of recombinant human IL-7suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15, and includes all forms ofIL-2 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares βand γ signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). Theamino acid sequence of recombinant human IL-15 suitable for use in theinvention is given in Table 2 (SEQ ID NO:7).

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21, and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4+ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human IL-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

When “an anti-tumor effective amount”, “an tumor-inhibiting effectiveamount”, or “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the genetically modified cytotoxiclymphocytes described herein may be administered at a dosage of 10⁴ to10¹¹ cells/kg body weight (e.g., 10⁵ to 10⁶, 10⁵ to 10¹⁰, 10⁵ to 10¹¹,10⁶ to 10¹¹, 10⁷ to 10¹¹, to 10¹⁰, to 10¹¹, 10⁶ to 10¹⁰, 10⁷ 10⁸ 10⁸ to10¹⁰, 10⁹ to 10¹¹, or 10⁹ to 10¹⁰ cells/kg body weight), including allinteger values within those ranges. Genetically modified cytotoxiclymphocytes compositions may also be administered multiple times atthese dosages. The genetically modified cytotoxic lymphocytes can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med 319:1676, 1988). The optimal dosage and treatment regime for a particularpatient can readily be determined by one skilled in the art of medicineby monitoring the patient for signs of disease and adjusting thetreatment accordingly.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer refers to malignant, neoplastic,or cancerous solid tumors. Solid tumor cancers include, but are notlimited to, sarcomas, carcinomas, and lymphomas, such as cancers of thelung, breast, prostate, colon, rectum, and bladder. The tissue structureof solid tumors includes interdependent tissue compartments includingthe parenchyma (cancer cells) and the supporting stromal cells in whichthe cancer cells are dispersed and which may provide a supportingmicroenvironment.

The term “liquid tumor” refers to an abnormal mass of cells that isfluid in nature. Liquid tumor cancers include, but are not limited to,leukemias, myelomas, and lymphomas, as well as other hematologicalmalignancies. TILs obtained from liquid tumors may also be referred toherein as marrow infiltrating lymphocytes (MILs).

The term “microenvironment,” as used herein, may refer to the solid orhematological tumor microenvironment as a whole or to an individualsubset of cells within the microenvironment. The tumor microenvironment,as used herein, refers to a complex mixture of “cells, soluble factors,signaling molecules, extracellular matrices, and mechanical cues thatpromote neoplastic transformation, support tumor growth and invasion,protect the tumor from host immunity, foster therapeutic resistance, andprovide niches for dominant metastases to thrive,” as described inSwartz, et al., Cancer Res., 2012, 72, 2473. Although tumors expressantigens that should be recognized by T cells, tumor clearance by theimmune system is rare because of immune suppression by themicroenvironment.

In an embodiment, the invention includes a method of treating a cancerwith a population of rTILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of rTILs accordingto the invention. In some embodiments, the population of rTILs may beprovided with a population of eTils, wherein a patient is pre-treatedwith nonmyeloablative chemotherapy prior to an infusion of rTILs andeTils according to the invention. In an embodiment, thenon-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days(days 27 and 26 prior to rTIL infusion) and fludarabine 25 mg/m2/d for 5days (days 27 to 23 prior to rTIL infusion). In an embodiment, afternon-myeloablative chemotherapy and rTIL infusion (at day 0) according tothe invention, the patient receives an intravenous infusion of IL-2intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (“cytokine sinks”). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the rTILs of the invention.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients (in a preferred embodiment of thepresent invention, for example, at least one potassium channel agonistin combination with a plurality of TILs) to a subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the subject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated (e.g., theweight, age and gender of the subject), the severity of the diseasecondition, or the manner of administration. The term also applies to adose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its developmentor progression; and (c) relieving the disease, i.e., causing regressionof the disease and/or relieving one or more disease symptoms.“Treatment” is also meant to encompass delivery of an agent in order toprovide for a pharmacologic effect, even in the absence of a disease orcondition. For example, “treatment” encompasses delivery of acomposition that can elicit an immune response or confer immunity in theabsence of a disease condition, e.g., in the case of a vaccine.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source, orcoding regions from different sources. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” (or synonyms thereof, e.g., “99% identical”) in the context oftwo or more nucleic acids or polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned (introducing gaps, if necessary) for maximumcorrespondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by particular alignmentsoftware. In certain embodiments, the default parameters of thealignment software are used.

As used herein, the term “variant” encompasses but is not limited toantibodies or fusion proteins which comprise an amino acid sequencewhich differs from the amino acid sequence of a reference antibody byway of one or more substitutions, deletions and/or additions at certainpositions within or adjacent to the amino acid sequence of the referenceantibody. The variant may comprise one or more conservativesubstitutions in its amino acid sequence as compared to the amino acidsequence of a reference antibody. Conservative substitutions mayinvolve, e.g., the substitution of similarly charged or uncharged aminoacids. The variant retains the ability to specifically bind to theantigen of the reference antibody. The term variant also includespegylated antibodies or proteins.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are outlined below.

III. Restimulation of Cryopreserved TILs

As discussed herein, the present invention relates to the restimulationof cryopreserved TILs to increase their metabolic activity and thusrelative health prior to transplant into a patient, and methods oftesting said metabolic health. As generally outlined herein, TILs aregenerally taken from a patient sample and manipulated to expand theirnumber prior to transplant into a patient. In some embodiments, the TILsmay be optionally genetically manipulated as discussed below, and thencryopreserved. Once thawed, they are then restimulated to increase theirmetabolism prior to infusion into a patient.

The “Step” Designations A, B, C, etc., below are in reference to FIG.11. The ordering of the Steps below and in FIG. 11 is exemplary and anycombination or order of steps, as well as additional steps, repetitionof steps, and/or omission of steps is contemplated by the presentapplication and the methods disclosed herein.

A. Step A: Obtain Patient Tumor Sample

In general, TILs are initially obtained from a patient tumor sample(“primary TILs”) and then expanded into a larger population for furthermanipulation as described herein, optionally cryopreserved, restimulatedas outlined herein and optionally evaluated for phenotype and metabolicparameters as an indication of TIL health.

A patient tumor sample may be obtained using methods known in the art,generally via surgical resection, needle biopsy or other means forobtaining a sample that contains a mixture of tumor and TIL cells. Ingeneral, the tumor sample may be from any solid tumor, including primarytumors, invasive tumors or metastatic tumors. The tumor sample may alsobe a liquid tumor, such as a tumor obtained from a hematologicalmalignancy. The solid tumor may be of any cancer type, including, butnot limited to, breast, pancreatic, prostate, colorectal, lung, brain,renal, stomach, and skin (including but not limited to squamous cellcarcinoma, basal cell carcinoma, and melanoma). In some embodiments,useful TILs are obtained from malignant melanoma tumors, as these havebeen reported to have particularly high levels of TILs.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer refers to malignant, neoplastic,or cancerous solid tumors. Solid tumor cancers include, but are notlimited to, sarcomas, carcinomas, and lymphomas, such as cancers of thelung, breast, triple negative breast cancer, prostate, colon, rectum,and bladder. In some embodiments, the cancer is selected from cervicalcancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma,pancreatic cancer, bladder cancer, breast cancer, triple negative breastcancer, and non-small cell lung carcinoma. The tissue structure of solidtumors includes interdependent tissue compartments including theparenchyma (cancer cells) and the supporting stromal cells in which thecancer cells are dispersed and which may provide a supportingmicroenvironment.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

Once obtained, the tumor sample is generally fragmented using sharpdissection into small pieces of between 1 to about 8 mm³, with fromabout 2-3 mm³ being particularly useful. The TILs are cultured fromthese fragments using enzymatic tumor digests. Such tumor digests may beproduced by incubation in enzymatic media (e.g., Roswell Park MemorialInstitute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanicaldissociation (e.g., using a tissue dissociator). Tumor digests may beproduced by placing the tumor in enzymatic media and mechanicallydissociating the tumor for approximately 1 minute, followed byincubation for 30 minutes at 37° C. in 5% CO₂, followed by repeatedcycles of mechanical dissociation and incubation under the foregoingconditions until only small tissue pieces are present. At the end ofthis process, if the cell suspension contains a large number of redblood cells or dead cells, a density gradient separation using FICOLLbranched hydrophilic polysaccharide may be performed to remove thesecells. Alternative methods known in the art may be used, such as thosedescribed in U.S. Patent Application Publication No. 2012/0244133 A1,the disclosure of which is incorporated by reference herein. Any of theforegoing methods may be used in any of the embodiments described hereinfor methods of expanding TILs or methods treating a cancer.

In some embodiments, fragmentation includes physical fragmentation,including for example, dissection as well as digestion. In someembodiments, the fragmentation is physical fragmentation. In someembodiments, the fragmentation is dissection. In some embodiments, thefragmentation is by digestion. In some embodiments, TILs can beinitially cultured from enzymatic tumor digests and tumor fragmentsobtained from patients.

In some embodiments, where the tumor is a solid tumor, the tumorundergoes physical fragmentation after the tumor sample is obtained, forexample such as in Step A of FIG. 11. In some embodiments, thefragmentation occurs before cryopreservation. In some embodiments, thefragmentation occurs after cryopreservation. In some embodiments, thefragmentation occurs after obtaining the tumor and in the absence of anycryopreservation. In some embodiments, the tumor is fragmented and 2, 3,or 4 fragments or pieces are placed in each container for the firstexpansion. In some embodiments, the tumor is fragmented and 3 or 4fragments or pieces are placed in each container for the firstexpansion. In some embodiments, the tumor is fragmented and 4 fragmentsor pieces are placed in each container for the first expansion,

In some embodiments, the TILs are obtained from tumor fragments. In someembodiments, the tumor fragment is obtained sharp dissection. In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³. Insome embodiments, the tumor fragment is between about 1 mm³ and 8 mm³.In some embodiments, the tumor fragment is about 1 mm³. In someembodiments, the tumor fragment is about 2 mm³. In some embodiments, thetumor fragment is about 3 mm³. In some embodiments, the tumor fragmentis about 4 mm³. In some embodiments, the tumor fragment is about 5 mm³.In some embodiments, the tumor fragment is about 6 mm³. In someembodiments, the tumor fragment is about 7 mm³. In some embodiments, thetumor fragment is about 8 mm³. In some embodiments, the tumor fragmentis about 9 mm³. In some embodiments, the tumor fragment is about 10 mm³.

In some embodiments, the TILs are obtained from tumor digests. In someembodiments, tumor digests were generated by incubation in enzyme media,for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mLgentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed bymechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, Calif.).After placing the tumor in enzyme media, the tumor can be mechanicallydissociated for approximately 1 minute. The solution can then beincubated for 30 minutes at 37° C. in 5% CO₂ and it then mechanicallydisrupted again for approximately 1 minute. After being incubated againfor 30 minutes at 37° C. in 5% CO₂, the tumor can be mechanicallydisrupted a third time for approximately 1 minute. In some embodiments,after the third mechanical disruption if large pieces of tissue werepresent, 1 or 2 additional mechanical dissociations were applied to thesample, with or without 30 additional minutes of incubation at 37° C. in5% CO₂. In some embodiments, at the end of the final incubation if thecell suspension contained a large number of red blood cells or deadcells, a density gradient separation using Ficoll can be performed toremove these cells.

In some embodiments, the harvested cell suspension prior to the firstexpansion step is called a “primary cell population” or a “freshlyharvested” cell population.

In some embodiments, cells can be optionally frozen after sample harvestand stored frozen prior to entry into Step B, which is described infurther detail below.

B. Step B: First Expansion

In some embodiments, a first expansion of TILs (also referred to as afirst expansion or first TIL expansion) may be performed using aninitial bulk TIL expansion step (for example, Step B as indicated inFIG. 11 or a first expansion step; this can include an expansion stepreferred to as preREP) as described below and herein, followed by asecond expansion step (for example, Step D as indicated in FIG. 11;which can include as an example what is referred to as a rapid expansionprotocol (REP) step) as described below and herein, followed by optionalcryopreservation (for example, after Step D as indicated in FIG. 11),and followed by an additional second expansion (for example, a secondStep D, as indicated in FIG. 11, which can include what is sometimesreferred to as a restimulation REP step) as described below and herein.The TILs obtained from this process may be optionally characterized forphenotypic characteristics and metabolic parameters as described herein.In some embodiments, the TILs are frozen (i.e., cryopreserved) after thefirst expansion (for example, Step B as indicated in FIG. 11) and storeduntil phenotyped for selection then thawed prior to proceeding to one ormore second expansion steps (for example, one or more expansionaccording to Step D as indicated in FIG. 11).

In some embodiments, where the cells are frozen after obtained from thetumor sample (such as, for example, during in Step A as indicated inFIG. 11), the cells are thawed prior to the first expansion (forexample, Step B as indicated in FIG. 11).

In embodiments where TIL cultures are initiated in 24-well plates, forexample, using Costar 24-well cell culture cluster, flat bottom (CorningIncorporated, Corning, N.Y., each well can be seeded with 1×10⁶ tumordigest cells or one tumor fragment in 2 mL of complete medium (CM) withIL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³.

After preparation of the tumor fragments, the resulting cells (i.e.,fragments) are cultured in serum containing IL-2 under conditions thatfavor the growth of TILs over tumor and other cells. In someembodiments, the tumor digests are incubated in 2 mL wells in mediacomprising inactivated human AB serum (or, in some cases, as outlinedherein, in the presence of aAPC cell population) with 6000 IU/mL ofIL-2. This primary cell population is cultured for a period of days,generally from 10 to 14 days, resulting in a bulk TIL population,generally about 1×10⁸ bulk TIL cells. In some embodiments, the growthmedia during the first expansion comprises IL-2 or a variant thereof. Insome embodiments, the IL is recombinant human IL-2 (rhIL-2). In someembodiments the IL-2 stock solution has a specific activity of 20-30×10⁶IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has aspecific activity of 20-×10⁶ IU/mg for a 1 mg vial. In some embodimentsthe IL-2 stock solution has a specific activity of 25×10⁶ IU/mg for a 1mg vial. In some embodiments the IL-2 stock solution has a specificactivity of 30×10⁶ IU/mg for a 1 mg vial. In some embodiments, the IL-2stock solution has a final concentration of 4-8×10⁶ IU/mg of IL-2. Insome embodiments, the IL-2 stock solution has a final concentration of5-7×10⁶ IU/mg of IL-2. In some embodiments, the IL-2 stock solution hasa final concentration of 6×10⁶ IU/mg of IL-2. In some embodiments, theIL-2 stock solution is prepare as described in Example 4. In someembodiments, first expansion culture media comprises about 10,000 IU/mLof IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2, about7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL ofIL-2. In some embodiments, first expansion culture media comprises about9,000 IU/mL of IL-2, to about 5,000 IU/mL of IL-2. In some embodiments,first expansion culture media comprises about 8,000 IU/mL of IL-2, toabout 6,000 IU/mL of IL-2. In some embodiments, first expansion culturemedia comprises about 7,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2.In some embodiments, first expansion culture media comprises about 6,000IU/mL of IL-2. In an embodiment, the cell culture medium furthercomprises IL-2. In some embodiments, the cell culture medium comprisesabout 3000 IU/mL of IL-2. In an embodiment, the cell culture mediumcomprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL ofIL-2. In an embodiment, the cell culture medium comprises between 1000and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mLof IL-2.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, it isreferred to as CM1 (culture medium 1). In some embodiments, CM consistsof RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mMHepes, and 10 mg/mL gentamicin. In embodiments where cultures areinitiated in gas-permeable flasks with a 40 mL capacity and a 10 cm²gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1), each flask was loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media was removed and replaced with fresh CM andIL-2 and after day 5, half the media was changed every 2-3 days. In someembodiments, the CM is the CM1 described in the Examples, see, Example5. In some embodiments, the first expansion occurs in an initial cellculture medium or a first cell culture medium. In some embodiments, theinitial cell culture medium or the first cell culture medium comprisesIL-2.

In some embodiments, the first TIL expansion can proceed for 11 days, 12days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20days, or 21 days. In some embodiments, the first TIL expansion canproceed for 11 days to 21 days. In some embodiments, the first TILexpansion can proceed for 12 days to 21 days. In some embodiments, thefirst TIL expansion can proceed for 13 days to 21 days. In someembodiments, the first TIL expansion can proceed for 14 days to 21 days.In some embodiments, the first TIL expansion can proceed for 15 days to21 days. In some embodiments, the first TIL expansion can proceed for 16days to 21 days. In some embodiments, the first TIL expansion canproceed for 17 days to 21 days. In some embodiments, the first TILexpansion can proceed for 18 days to 21 days. In some embodiments, thefirst TIL expansion can proceed for 19 days to 21 days. In someembodiments, the first TIL expansion can proceed for 20 days to 21 days.In some embodiments, the first TIL expansion can proceed for 21 days.

C. Step C: First Expansion to Second Expansion Transition

In some embodiments, the TILs obtained from the first expansion (forexample, from Step B as indicated in FIG. 11) are stored untilphenotyped for selection. In some embodiments, the TILs obtained fromthe first expansion are cryopreserved after the first expansion andprior to the second expansion. In some embodiments, the TILs arecryopreserved as part of the first expansion to second expansiontransition. For example, in some embodiments, the TILs are cryopreservedafter Step B and before Step D as indicated in FIG. 11. In someembodiments, the TILs are cryopreserved and thawed as part of the firstexpansion to second expansion transition. For example, in someembodiments, the TILs are cryopreserved after Step B then thawed priorto proceeding to Step D (as provided in FIG. 11). In some embodiments,the transition from the first expansion to the second expansion occursat about 22 days, 23, days, 24 days, 25 days, 26 days, 27 days, 28 days,29 days, or 30 days from when fragmentation occurs. In some embodiments,the transition from the first expansion to the second expansion occursat about 22 days to 30 days from when fragmentation occurs. In someembodiments, the transition from the first expansion to the secondexpansion occurs at about 24 days to 30 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs at about 26 days to 30 days from whenfragmentation occurs. In some embodiments, the transition from the firstexpansion to the second expansion occurs at about 28 days to 30 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs at about 30 days fromwhen fragmentation occurs.

D. Step D: Second Expansion

In some embodiments, the second expansion or second TIL expansion (whichcan include expansions sometimes referred to as REP) of TIL can beperformed using any TIL flasks or containers known by those of skill inthe art. In some embodiments, the second TIL expansion can proceed for14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days,or 22 days. In some embodiments, the second TIL expansion can proceedfor about 14 days to about 22 days. In some embodiments, the second TILexpansion can proceed for about 14 days to about 20 days. In someembodiments, the second TIL expansion can proceed for about 14 days toabout 18 days. In some embodiments, the second TIL expansion can proceedfor about 14 days to about 16 days. In some embodiments, the second TILexpansion can proceed for about 14 days.

In some embodiments, the second expansion occurs in a supplemented cellculture medium. In some embodiments, the supplemented cell culturemedium comprises IL-2, OKT-3, and antigen-presenting feeder cells. Insome embodiments, the second cell culture medium comprises IL-2, OKT-3,and antigen-presenting cells (APCs; also referred to asantigen-presenting feeder cells).

In some embodiments, the second expansion (which can include expansionsreferred to as REP) of TILs can be performed using T-175 flasks andgas-permeable bags as previously described (Tran K Q, Zhou J, DurflingerK H, et al., 2008, J Immunother., 31:742-751, and Dudley M E, WunderlichJ R, Shelton T E, et al. 2003, J Immunother., 26:332-342) orgas-permeable G-Rex flasks. In some embodiments, the second expansion isperformed using flasks. In some embodiments, the second expansion isperformed using gas-permeable G-Rex flasks. For TIL the second expansionin T-175 flasks, about 1×10⁶ TIL are suspended in about 150 mL of mediaand this is added to each T-175 flask. The TIL are cultured withirradiated (50 Gy) allogeneic PBMC as “feeder” cells at a ratio of 1 to100 and the cells were cultured in a 1 to 1 mixture of CM and AIM-Vmedium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mLof anti-CD3. The T-175 flasks are incubated at 37° C. in 5% CO₂. In someembodiments, half the media is changed 5 days into the second expansionusing 50/50 medium with 3000 IU/mL of IL-2. In some embodiments, on day7, cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL ofAIM-V with 5% human AB serum and 3000 IU/mL of IL-2 is added to the 300mL of TIL suspension. The number of cells in each bag can be countedevery day or two and fresh media can be added to keep the cell countbetween about 0.5 and about 2.0×10⁶ cells/mL.

In some embodiments, the second expansion (which can include expansionsreferred to as REP) of TIL can be performed in 500 mL capacity gaspermeable flasks with 100 cm² gas-permeable silicon bottoms (G-Rex 100,commercially available from Wilson Wolf Manufacturing Corporation, NewBrighton, Minn., USA) (FIG. 1), about 5×10⁶ or 10×10⁶ TIL are culturedwith irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30 ng/mL ofanti-CD3 (OKT3). The G-Rex100 flasks can be incubated at 37° C. in 5%CO₂. In some embodiments, 5 days into the second expansion, 250 mL ofsupernatant is removed and placed into centrifuge bottles andcentrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellets can thenbe resuspended with 150 mL of fresh medium with 5% human AB serum, 3000IU per mL of IL-2 and added back to the original G-Rex100 flasks. Inembodiments where TILs are expanded serially in G-Rex100 flasks, on day7 the TIL in each G-Rex100 are suspended in the 300 mL of media presentin each flask and the cell suspension was divided into three 100 mLaliquots that can be used to seed three G-Rex100 flasks. Then 150 mL ofAIM-V with 5% human AB serum and 3000 IU per mL of IL-2 can be added toeach flask. The G-Rex100 flasks can be incubated at 37° C. in 5% CO₂ andafter 4 days in to the second expansion, 150 mL of AIM-V with 3000 IUper mL of IL-2 can be added to each G-Rex100 flask. In some embodiments,the cells are harvested on day 14 of culture.

In some embodiments, the second expansion (which can include expansionsreferred to as REP) of TIL can be performed in a gas permeablecontainer. For example, TILs can be rapidly expanded using non-specificT-cell receptor stimulation in the presence of interleukin-2 (IL-2) orinterleukin-15 (IL-15). In an embodiment, expansion of the number ofTILs uses about 1×10⁹ to about 1×10¹¹ antigen-presenting feeder cells.The non-specific T-cell receptor stimulus can include, for example,about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody(commercially available from Ortho-McNeil, Raritan, N.J. or MiltenyiBiotech, Auburn, Calif.). TILs can be rapidly expanded furtherstimulation of the TILs in vitro with one or more antigens, includingantigenic portions thereof, such as epitope(s), of the cancer, which canbe optionally expressed from a vector, such as a human leukocyte antigenA2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl00:209-217 (210M), optionally in the presence of a T-cell growth factor,such as 300 IU/mL IL-2 or IL-15. Other suitable antigens may include,e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2,and VEGFR2, or antigenic portions thereof. TIL may also be rapidlyexpanded by re-stimulation with the same antigen(s) of the cancer pulsedonto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILscan be further restimulated with, e.g., example, irradiated, autologouslymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the second expansion (which can include expansionsreferred to as REP) of TIL can be performed in 500 mL capacity gaspermeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100,commercially available from Wilson Wolf Manufacturing Corporation, NewBrighton, Minn., USA), 5×10⁶ or 10×10⁶ TIL may be cultured with aAPCs ata ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 5%human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3(OKT3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO₂. Onday 5, 250 mL of supernatant may be removed and placed into centrifugebottles and centrifuged at 1500 rpm (491×g) for 10 minutes. The TILpellets may be re-suspended with 150 mL of fresh medium with 5% human ABserum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100flasks. When TIL are expanded serially in G-Rex 100 flasks, on day 7 theTIL in each G-Rex 100 may be suspended in the 300 mL of media present ineach flask and the cell suspension may be divided into 3 100 mL aliquotsthat may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask.The G-Rex 100 flasks may be incubated at 37° C. in 5% CO₂ and after 4days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to eachG-Rex100 flask. The cells may be harvested on day 14 of culture.

In one embodiment, the second expansion (including expansions referredto as REP) is performed in flasks with the bulk TILs being mixed with a100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media. Media replacementis done (generally 2/3 media replacement via respiration with freshmedia) until the cells are transferred to an alternative growth chamber.Alternative growth chambers include GRex flasks and gas permeablecontainers as more fully discussed below.

In another embodiment, the second expansion (including expansionsreferred to as REP) is performed and further comprises a step whereinTILs are selected for superior tumor reactivity. Any selection methodknown in the art may be used. For example, the methods described in U.S.Patent Application Publication No. 2016/0010058 A1, the disclosures ofwhich are incorporated herein by reference, may be used for selection ofTILs for superior tumor reactivity.

Optionally, a cell viability assay can be performed after the secondexpansion (including expansions referred to as the REP expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. In some embodiments, TILsamples can be counted and viability determined using a Cellometer K2automated cell counter (Nexcelom Bioscience, Lawrence, Mass.). In someembodiments, viability is determined according to the Cellometer K2Image Cytometer Automatic Cell Counter protocol described, for example,in Example 2.

In some embodiments, cells are grown for 7 days, 8 days, 9 days, 10days, or 11 days of the total second expansion time before being splitinto more than one container or flask.

In some embodiments, the second expansion culture medium (e.g.,sometimes referred to as CM2 or the second cell culture medium),comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells(APCs), as discussed in more detail below.

In some embodiments, the antigen-presenting feeder cells are PBMCs. Insome embodiments, the antigen-presenting feeder cells are artificialantigen-presenting feeder cells. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is about 1 to25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375,about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILsto antigen-presenting feeder cells in the second expansion is between 1to 50 and 1 to 300. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is between 1 to100 and 1 to 200.

In an embodiment, the TIL expansion procedures described herein requirean excess of feeder cells during the second expansion (including forexample, expansions referred to as REP TIL expansions). In manyembodiments, the feeder cells are peripheral blood mononuclear cells(PBMCs) obtained from standard whole blood units from healthy blooddonors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation. In an embodiment, artificialantigen-presenting (aAPC) cells are used in place of PBMCs.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the REP procedures.

In some embodiments, the growth media during the first expansioncomprises IL-2 or a variant thereof. In some embodiments, the IL isrecombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stocksolution has a specific activity of 20-30×10⁶ IU/mg for a 1 mg vial. Insome embodiments the IL-2 stock solution has a specific activity of20-×10⁶ IU/mg for a 1 mg vial. In some embodiments the IL-2 stocksolution has a specific activity of 25×10⁶ IU/mg for a 1 mg vial. Insome embodiments the IL-2 stock solution has a specific activity of30×10⁶ IU/mg for a 1 mg vial. In some embodiments, the IL-2 stocksolution has a final concentration of 4-8×10⁶ IU/mg of IL-2. In someembodiments, the IL-2 stock solution has a final concentration of5-7×10⁶ IU/mg of IL-2. In some embodiments, the IL-2 stock solution hasa final concentration of 6×10⁶ IU/mg of IL-2. In some embodiments, theIL-2 stock solution is prepare as described in Example 4. In someembodiments, first expansion culture media comprises about 10,000 IU/mLof IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2, about7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL ofIL-2. In some embodiments, first expansion culture media comprises about9,000 IU/mL of IL-2, to about 5,000 IU/mL of IL-2. In some embodiments,first expansion culture media comprises about 8,000 IU/mL of IL-2, toabout 6,000 IU/mL of IL-2. In some embodiments, first expansion culturemedia comprises about 7,000 IU/mL of IL-2, to about 6,000 IU/mL of IL-2.In some embodiments, first expansion culture media comprises about 6,000IU/mL of IL-2. In an embodiment, the cell culture medium furthercomprises IL-2. In some embodiments, the cell culture medium comprisesabout 3000 IU/mL of IL-2. In an embodiment, the cell culture mediumcomprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL ofIL-2. In an embodiment, the cell culture medium comprises between 1000and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mLof IL-2.

In some embodiments, the second expansion cell culture media alsoincludes an anti-CD3 antibody. In some embodiment, the cell culturemedium comprises OKT3 antibody. In some embodiments, the cell culturemedium comprises about 30 ng/mL of OKT3 antibody. In an embodiment, thecell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL,about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL,about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about500 ng/mL, and about 1 μg/mL of OKT3 antibody. In an embodiment, thecell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL,between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL ofOKT3 antibody.

In some embodiments, an anti-CD3 antibody in combination with IL-2induces T cell activation and cell division in the TIL population. Thiseffect can be seen with full length antibodies as well as Fab andF(ab′)2 fragments, with the former being generally preferred; see, e.g.,Tsoukas et al., J. Immunol. 1985, 135, 1719, hereby incorporated byreference in its entirety. As will be appreciated by those in the art,there are a number of suitable anti-human CD3 antibodies that find usein the invention, including anti-human CD3 polyclonal and monoclonalantibodies from various mammals, including, but not limited to, murine,human, primate, rat, and canine antibodies. In particular embodiments,the OKT3 anti-CD3 antibody is used (commercially available fromOrtho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif.).

In some embodiment, the cells in the second expansion are grown in aculture media with high doses of a cytokine, in particular IL-2, as isknown in the art.

Alternatively, using combinations of cytokines for the second expansionof TILS is additionally possible, with combinations of two or more ofIL-2, IL-15 and IL-21 as is generally outlined in InternationalPublication No. WO 2015/189356 and International Publication No. WO2015/189357, hereby expressly incorporated by reference in theirentirety. Thus, possible combinations include IL-2 and IL-15, IL-2 andIL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latterfinding particular use in many embodiments. The use of combinations ofcytokines specifically favors the generation of lymphocytes, and inparticular T-cells as described therein.

E. Optional Repeats of Step D: Second Expansion

In some embodiments, the second expansion is performed one or moretimes, i.e., the second expansion is repeated. For example, in someembodiments the Step D second expansion as indicated in FIG. 11 isrepeated one or more times. In some embodiments, the second expansion isreferred to as an additional second expansion. In some embodiments wherethe second expansion is performed more than once (i.e., where the secondexpansion is repeated), this can include procedures referred to as a TILRapid Expansion Protocol. In some embodiments, the TIL cell populationis expanded in number after harvest and first expansion. This process isgenerally referred to in the art as a rapid expansion process (REP) andthe repeated second expansion can include expansion referred to asreREP. This overall protocol can be generally accomplished using culturemedia comprising a number of components, including feeder cells, acytokine source, and an anti-CD3 antibody, in a gas-permeable container.In some embodiments, one or more subsequent second expansion(s) areperformed as described above. In some embodiments, one or moresubsequent second expansions are performed as provided in under Step Din FIG. 11 and prior to Step E as provide in FIG. 11. In someembodiments, one, two, three, four or more second expansions areperformed as described above. In some embodiments, one, two, three, fouror more second expansions are performed as provided in Step D of FIG. 11before Step E of FIG. 11. In some embodiments, two second expansions areperformed as described above. In some embodiments, two second expansionsare performed as provided in Step D of FIG. 11 before Step E of FIG. 11.In some embodiments, three second expansions are performed as describedabove. In some embodiments, three second expansions are performed asprovided in Step D of FIG. 11 before Step E of FIG. 11. In someembodiments, four second expansions are performed as described above. Insome embodiments, four second expansions are performed as provided inStep D of FIG. 11 before Step E of FIG. 11.

In some embodiments, the repeat of the second expansion of the TILS(such as for example in Step D of FIG. 11) can be referred to as arestimulation of TILs. In some embodiments, the present inventionincludes a restimulation step, i.e., a repeat of the second expansion(for example, a repeat of the second expansion from Step D of FIG. 11).In some embodiments, the repeated second expansion (which can include anexpansion referred to as a restimulation step (“reREP”)) is performed oncells that have been cryopreserved. In some embodiments, the TILs arecryopreserved after Step D. In some embodiments, after an initial secondexpansion in Step D, the cells may be cultured in regular media, e.g. a“resting” media, and then one or more second expansions steps areperformed. In some embodiments, the resting media comprises IL-2. Insome embodiments, the resting media does not comprise IL-2. In someembodiments, the resting media is a standard cell culture media known inthe art. In some embodiments, the resting media is AIM-V, DMEM,DMEM/F12, MEM, RPMI, OptiMEM, IMDM, or any other standard media that isknown in art, including commercially available media. In someembodiments, the resting media is AIM-V.

In general, as discussed herein, the TILs are initially prepared byobtaining a primary population of TILs from a tumor resected from apatient as discussed herein (the “primary cell population” or “firstcell population”). This is followed with an initial bulk expansionutilizing a culturing of the cells with IL-2, forming a secondpopulation of cells (sometimes referred to herein as the “bulk TILpopulation” or “second population”). In some embodiments, this is alsoreferred to as the initial or first expansion.

The bulk TIL population (for example, the population obtained from forexample Step A in FIG. 11) is then subjected to a REP step, sometimesreferred to as a first expansion (for example, the first expansion asdescribed in Step B of FIG. 11) in a cell culture media comprising IL-2,OKT-3, and antigen presenting feeder cells (APCs), wherein the APCsgenerally comprise peripheral blood mononuclear cells (PBMCs; or,alternatively as discussed herein, using antigen presenting cells),wherein the rapid expansion (for example, the second expansion asprovide in Step D of FIG. 11) is performed for at least 14 days. Asdiscussed herein, the media may also contain combinations of IL-2, IL-15and/or IL-23 rather than IL-2 alone. In some embodiments, this postsecond expansion (for example, post Step D of FIG. 11) expanded TILpopulation is at least 50-fold or 100-fold greater in number than thesecond population of TILs (for example, the population of TILs obtainedfrom Step B of FIG. 11). In some embodiments, the population of TILsobtained after the second expansion in Step D of FIG. 11 are 50-fold or100-fold greater in number than the TILs obtained from the firstexpansion in Step B of FIG. 11. TILs are measured by cell countingmethods known in the art, including those methods described in theExamples provided herewith, including Examples 1, 2, and 3. In someembodiments, a K2 cell counter is employed to count the TILs. In someembodiments, a Cellometer IC2 Image cytometer is employed to count theTILs.

In some embodiments, as discussed herein, the TIL population obtainedafter the second expansion (sometimes referred to as a third TILpopulation or a REP cell population) is removed from the supplementedcell culture media (for example, the culture media used in Step D ofFIG. 11 or the media referred to as CM2 in the Examples) and optionallycryopreserved in a storage media (for example, media containing 5% DMSO)prior to performing and additional second expansion step.

Optionally, the TILs can be cryopreserved after a second expansion andbefore an additional second expansion. In some embodiments, the TILs arecryopreserved after performing Step D of FIG. 11 and before performingan additional Step D of FIG. 11. In some embodiments, the cryopreservedTILs are thawed prior to performing the additional second expansion. Insome embodiments, the cryopreserved TILs are thawed prior to performingthe additional Step D as provided in FIG. 11. In some embodiments, theTILs are cryopreserved in 5% DMSO. In some embodiments, the TILs arecryopreserved in cell culture media plus 5% DMSO. Alternatively, thecells are removed from the supplemented cell culture media (for example,the culture media used in Step D of FIG. 11) and cultured in a restingmedia. Such media include those that are described in Examples 1 and 5,as well as the other Examples provided herewith. In some embodiments,resting media can include media with IL-2. In some embodiments, theresting media can be the media referred to as CM1 in the examples.

The additional second expansion (including expansions referred to asreREP) is done on either the thawed cells or resting cells, using asupplemented cell culture medium (for example, a medium as provide inStep D of FIG. 11) comprising IL-2, OKT-3, and feeder cells (forexample, antigen presenting cells), generally comprising peripheralblood mononuclear cells (PBMCs; or, alternatively as discussed herein,using antigen presenting cells), wherein the additional second expansionis performed for at least 14 days. As discussed herein, the media mayalso contain combinations of IL-2, IL-15 and/or IL-23 rather than IL-2alone.

This results in an expanded population of TILs that are characterized inthat these expanded TILs exhibits an increased subpopulation of effectorT cells and/or central memory T cells relative to the second populationof TILs (e.g., the bulk starting TILs). In some embodiments, theseexpanded TILs are the TILs obtained from Step D of FIG. 11.

In some embodiments the memory T cells are those cells thatconstitutively CCR7 and CD62L. See, Sallusto, et al., Annu. Rev.Immunol., 2004, 22:745-763; incorporated by reference herein in itsentirety.

Thus, the present invention provides methods for the restimulation ofcryopreserved TILs upon thawing, based on post-thaw methods that resultin increases of metabolic health such as glycolysis and respiration. Insome embodiments, method comprises providing a population of thawedcryopreserved TILs that are then treated to increase their metabolichealth to allow for optimal treatment upon infusion into patients.

F. Step E: Harvest TILS from Step D

After the second expansion step, cells can be harvested. In someembodiments the TILs are harvested after one, two, three, four or moresecond expansion steps. In some embodiments, the TILs are harvestedafter one, two, three, four or more second expansion steps according toStep D as provided in FIG. 11.

TILs can be harvested in any appropriate and sterile manner, includingfor example by centrifugation. Methods for TIL harvesting are well knownin the art and any such know methods can be employed with the presentprocess.

G. Step F: Final Formulation and/or Transfer to Infusion Bag

After Steps A through E as provided in an exemplary order in FIG. 11 andas outlined in detailed above and herein are complete, cells aretransferred to a container for use in administration to a patient. Insome embodiments, once a therapeutically sufficient number of TILs areobtained using the expansion methods described above, they aretransferred to a container for use in administration to a patient.

In an embodiment, TILs expanded using APCs of the present disclosure areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using PBMCs of the present disclosure maybe administered by any suitable route as known in the art. In someembodiments, the T-cells are administered as a single intra-arterial orintravenous infusion, which preferably lasts approximately 30 to 60minutes. Other suitable routes of administration includeintraperitoneal, intrathecal, and intralymphatic.

1. Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, TILs expanded using APCs of the present disclosure areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using PBMCs of the present disclosure maybe administered by any suitable route as known in the art. In someembodiments, the T-cells are administered as a single intra-arterial orintravenous infusion, which preferably lasts approximately 30 to 60minutes. Other suitable routes of administration includeintraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. In some embodiments, atherapeutically sufficient number of TILs are needed for a suitabledosage. In some embodiments, from about 2.3×10¹⁰ to about 13.7×10¹⁰ TILsare administered, with an average of around 7.8×10¹⁰ TILs, particularlyif the cancer is melanoma. In an embodiment, about 1.2×10¹⁰ to about4.3×10¹⁰ of TILs are administered. In some embodiments, about 3×10¹⁰ toabout 12×10¹⁰ TILs are administered. In some embodiments, about 4×10¹⁰to about 10×10¹⁰ TILs are administered. In some embodiments, about5×10¹⁰ to about 8×10¹⁰ TILs are administered. In some embodiments, about6×10¹⁰ to about 8×10¹⁰ TILs are administered. In some embodiments, about7×10¹⁰ to about 8×10¹⁰ TILs are administered. In some embodiments, thetherapeutically effective dosage is about 2.3×10¹⁰ to about 13.7×10¹⁰.In some embodiments, the therapeutically effective dosage is about7.8×10¹⁰ TILs, particularly of the cancer is melanoma. In someembodiments, the therapeutically effective dosage is about 1.2×10¹⁰ toabout 4.3×10¹⁰ of TILs. In some embodiments, the therapeuticallyeffective dosage is about 3×10¹⁰ to about 12×10¹⁰ TILs. In someembodiments, the therapeutically effective dosage is about 4×10¹⁰ toabout 10×10¹⁰ TILs. In some embodiments, the therapeutically effectivedosage is about 5×10¹⁰ to about 8×10¹⁰ TILs. In some embodiments, thetherapeutically effective dosage is about 6×10¹⁰ to about 8×10¹⁰ TILs.In some embodiments, the therapeutically effective dosage is about7×10¹⁰ to about 8×10¹⁰ TILs.

In some embodiments, the number of the TILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the number of theTILs provided in the pharmaceutical compositions of the invention is inthe range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰,1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹²,and 5×10¹² to 1×10¹³. In some embodiments, the therapeutically effectivedosage is about 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶,9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷,1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹,2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰,3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹,3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹²,3×10¹², 4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³,3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%,8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%,5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%,2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9%to about 12% or about 1% to about 10% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%,about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% toabout 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/vor v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

The TILs provided in the pharmaceutical compositions of the inventionare effective over a wide dosage range. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of theTILs may also be used if appropriate. The amounts of the pharmaceuticalcompositions administered using the methods herein, such as the dosagesof TILs, will be dependent on the human or mammal being treated, theseverity of the disorder or condition, the rate of administration, thedisposition of the active pharmaceutical ingredients and the discretionof the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of TILs maycontinue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, an effectivedosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to1×10¹²1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, including intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, topically, bytransplantation, or by inhalation.

H. Optional Cell Viability Analyses

Optionally, a cell viability assay can be performed after the Step Bfirst expansion, using standard assays known in the art. For example, atrypan blue exclusion assay can be done on a sample of the bulk TILs,which selectively labels dead cells and allows a viability assessment.Other assays for use in testing viability can include but are notlimited to the Alamar blue assay; and the MTT assay.

1. Cell Counts, Viability, Flow Cytometry

In some embodiments, cell counts and/or viability are measured. Theexpression of markers such as but not limited CD3, CD4, CD8, and CD56,as well as any other disclosed or described herein, can be measured byflow cytometry with antibodies, for example but not limited to thosecommercially available from BD Bio-sciences (BD Biosciences, San Jose,Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cellscan be counted manually using a disposable c-chip hemocytometer (VWR,Batavia, Ill.) and viability can be assessed using any method known inthe art, including but not limited to trypan blue staining.

In some cases, the bulk TIL population can be cryopreserved immediately,using the protocols discussed below. Alternatively, the bulk TILpopulation can be subjected to REP and then cryopreserved as discussedbelow. Similarly, in the case where genetically modified TILs will beused in therapy, the bulk or REP TIL populations can be subjected togenetic modifications for suitable treatments.

2. Cell Cultures

In an embodiment, a method for expanding TILs may include using about5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cellmedium. In an embodiment, expanding the number of TILs uses no more thanone type of cell culture medium. Any suitable cell culture medium may beused, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate,and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, CarlsbadCalif.). In this regard, the inventive methods advantageously reduce theamount of medium and the number of types of medium required to expandthe number of TIL. In an embodiment, expanding the number of TIL maycomprise adding fresh cell culture media to the cells (also referred toas feeding the cells) no more frequently than every third or fourth day.Expanding the number of cells in a gas permeable container simplifiesthe procedures necessary to expand the number of cells by reducing thefeeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium thereinusing aAPCs for a duration of about 14 to about 42 days, e.g., about 28days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L. In an embodiment, TILs can be expanded inG-Rex flasks (commercially available from Wilson Wolf Manufacturing).Such embodiments allow for cell populations to expand from about 5×10⁵cells/cm² to between 10×10⁶ and 30×10⁶ cells/cm². In an embodiment thisexpansion is conducted without adding fresh cell culture media to thecells (also referred to as feeding the cells). In an embodiment, this iswithout feeding so long as medium resides at a height of about 10 cm inthe GRex flask. In an embodiment this is without feeding but with theaddition of one or more cytokines. In an embodiment, the cytokine can beadded as a bolus without any need to mix the cytokine with the medium.Such containers, devices, and methods are known in the art and have beenused to expand TILs, and include those described in U.S. PatentApplication Publication No. US 2014/0377739A1, International PublicationNo. WO 2014/210036 A1, U.S. Patent Application Publication No. us2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S.Patent Application Publication No. US 2011/0136228 A1, U.S. Pat. No.8,809,050 B2, International publication No. WO 2011/072088 A2, U.S.Patent Application Publication No. US 2016/0208216 A1, U.S. PatentApplication Publication No. US 2012/0244133 A1, InternationalPublication No. WO 2012/129201 A1, U.S. Patent Application PublicationNo. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2, InternationalPublication No. WO 2013/173835 A1, U.S. Patent Application PublicationNo. US 2015/0175966 A1, the disclosures of which are incorporated hereinby reference. Such processes are also described in Jin et al., J.Immunotherapy, 2012, 35:283-292. Optional Genetic Engineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

I. Optional Cryopreservation of TILs

As discussed above in Steps A through E, cryopreservation can occur atnumerous points throughout the TIL expansion process. In someembodiments, the bulk TIL population after the first expansion accordingto Step B or the expanded population of TILs after the one or moresecond expansions according to Step D can be cryopreserved.Cryopreservation can be generally accomplished by placing the TILpopulation into a freezing solution, e.g., 85% complement inactivated ABserum and 15% dimethyl sulfoxide (DMSO). The cells in solution areplaced into cryogenic vials and stored for 24 hours at −80° C., withoptional transfer to gaseous nitrogen freezers for cryopreservation.See, Sadeghi, et al., Acta Oncologica 2013, 52, 978-986. In someembodiments, the TILs are cryopreserved in 5% DMSO. In some embodiments,the TILs are cryopreserved in cell culture media plus 5% DMSO. In someembodiments, the TILs are cryopreserved according to the methodsprovided in Examples 8 and 9.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately 4/5 of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

J. Phenotypic Characteristics of Expanded TILs

In some embodiment, the TILs are analyzed for expression of numerousphenotype markers after expansion, including those described herein andin the Examples. In an embodiment, expression of one or more phenotypicmarkers is examined. In some embodiments, the phenotypic characteristicsof the TILs are analyzed after the first expansion in Step B. In someembodiments, the phenotypic characteristics of the TILs are analyzedduring the transition in Step C. In some embodiments, the phenotypiccharacteristics of the TILs are analyzed during the transition accordingto Step C and after cryopreservation. In some embodiments, thephenotypic characteristics of the TILs are analyzed after the secondexpansion according to Step D. In some embodiments, the phenotypiccharacteristics of the TILs are analyzed after two or more expansionsaccording to Step D. In some embodiments, the marker is selected fromthe group consisting of TCRab, CD57, CD28, CD4, CD27, CD56, CD8a,CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, themarker is selected from the group consisting of TCRab, CD57, CD28, CD4,CD27, CD56, and CD8a. In an embodiment, the marker is selected from thegroup consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. Insome embodiments, expression of one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, or fourteen markers isexamined. In some embodiments, the expression from one or more markersfrom each group is examined. In some embodiments, one or more of HLA-DR,CD38, and CD69 expression is maintained (i.e., does not exhibit astatistically significant difference) in fresh TILs as compared tothawed TILs. In some embodiments, the activation status of TILs ismaintained in the thawed TILs.

In an embodiment, expression of one or more regulatory markers ismeasured. In some embodiments, the regulatory marker is selected fromthe group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, TIM-3, CD69,CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, theregulatory marker is selected from the group consisting of CD137, CD8a,Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatorymarker is selected from the group consisting of CD69, CD8a, TIGIT, CD4,CD3, KLRG1, and CD154. In some embodiments, regulatory moleculeexpression is decreased in thawed TILs as compared to fresh TILs. Insome embodiments, expression of regulatory molecules LAG-3 and TIM-3 isdecreased in thawed TILs as compared to fresh TILs. In some embodiments,there is no significant difference in CD4, CD8, NK, TCRαβ expression. Insome embodiments, there is no significant difference in CD4, CD8, NK,TCRαβ expression, and/or memory markers in fresh TILs as compared tothawed TILs.

In some embodiments the memory marker is selected from the groupconsisting of CCR7 and CD62L

In some embodiments, the viability of the fresh TILs as compared to thethawed TILs is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98%. In some embodiments, theviability of both the fresh and thawed TILs is greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, or greater than 98%. In some embodiments, the viability ofboth the fresh and thawed product is greater than 80%, greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,or greater than 90%. In some embodiments, the viability of both thefresh and thawed product is greater than 86%.

In an embodiment, restimulated TILs can also be evaluated for cytokinerelease, using cytokine release assays. In some embodiments, TILs can beevaluated for interferon-7 (IFN-7) secretion in response to stimulationeither with OKT3 or co-culture with autologous tumor digest. Forexample, in embodiments employing OKT3 stimulation, TILs are washedextensively, and duplicate wells are prepared with 1×10⁵ cells in 0.2 mLCM in 96-well flat-bottom plates precoated with 0.1 or 1.0 μg/mL of OKT3diluted in phosphate-buffered saline. After overnight incubation, thesupernatants are harvested and IFN-7 in the supernatant is measured byELISA (Pierce/Endogen, Woburn, Mass.). For the co-culture assay, 1×10⁵TIL cells are placed into a 96-well plate with autologous tumor cells.(1:1 ratio). After a 24-hour incubation, supernatants are harvested andIFN-7 release can be quantified, for example by ELISA.

Flow cytometric analysis of cell surface biomarkers: TIL samples werealiquoted for flow cytometric analysis of cell surface markers see, forExample see Examples 7, 8, and 9.

In some embodiments, the TILs are being evaluated for various regulatorymarkers. In some embodiments, the regulatory marker is selected from thegroup consisting of TCR α/β, CD56, CD27, CD28, CD57, CD45RA, CD45RO,CD25, CD127, CD95, IL-2R−, CCR7, CD62L, KLRG1, and CD122. In someembodiments, the regulatory marker is TCR α/β. In some embodiments, theregulatory marker is CD56. In some embodiments, the regulatory marker isCD27. In some embodiments, the regulatory marker is CD28. In someembodiments, the regulatory marker is CD57. In some embodiments, theregulatory marker is CD45RA. In some embodiments, the regulatory markeris CD45RO. In some embodiments, the regulatory marker is CD25. In someembodiments, the regulatory marker is CD127. In some embodiments, theregulatory marker is CD95. In some embodiments, the regulatory marker isIL-2R−. In some embodiments, the regulatory marker is CCR7. In someembodiments, the regulatory marker is CD62L. In some embodiments, theregulatory marker is KLRG1. In some embodiments, the regulatory markeris CD122.

K. Metabolic Health of Expanded TILs

The restimulated TILs are characterized by significant enhancement ofbasal glycolysis as compared to either freshly harvested TILs and/orpost-thawed TILs.

Spare respiratory capacity (SRC) and glycolytic reserve can be evaluatedfor TILs expanded with aEM3 aAPCs in comparison to PBMC feeders. TheSeahorse XF Cell Mito Stress Test measures mitochondrial function bydirectly measuring the oxygen consumption rate (OCR) of cells, usingmodulators of respiration that target components of the electrontransport chain in the mitochondria. The test compounds (oligomycin,FCCP, and a mix of rotenone and antimycin A, described below) areserially injected to measure ATP production, maximal respiration, andnon-mitochondrial respiration, respectively. Proton leak and sparerespiratory capacity are then calculated using these parameters andbasal respiration. Each modulator targets a specific component of theelectron transport chain. Oligomycin inhibits ATP synthase (complex V)and the decrease in OCR following injection of oligomycin correlates tothe mitochondrial respiration associated with cellular ATP production.Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is anuncoupling agent that collapses the proton gradient and disrupts themitochondrial membrane potential. As a result, electron flow through theelectron transport chain is uninhibited and oxygen is maximally consumedby complex IV. The FCCP-stimulated OCR can then be used to calculatespare respiratory capacity, defined as the difference between maximalrespiration and basal respiration. Spare respiratory capacity (SRC) is ameasure of the ability of the cell to respond to increased energydemand. The third injection is a mix of rotenone, a complex I inhibitor,and antimycin A, a complex III inhibitor. This combination shuts downmitochondrial respiration and enables the calculation ofnonmitochondrial respiration driven by processes outside themitochondria.

In some embodiments, the metabolic assay is basal respiration. Ingeneral, second expansion TILs or second additional expansion TILs (suchas, for example, those described in Step D of FIG. 11, including TILsreferred to as reREP TILs) have a basal respiration rate that is atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% of the basal respiration rateof freshly harvested TILs. In some embodiments, the basal respirationrate is from about 50% to about 99% of the basal respiration rate offreshly harvested TILs. In some embodiments, the basal respiration rateis from about 60% to about 99% of the basal respiration rate of freshlyharvested TILs. In some embodiments, the basal respiration rate is fromabout 70% to about 99% of the basal respiration rate of freshlyharvested TILs. In some embodiments, the basal respiration rate is fromabout 80% to about 99% of the basal respiration rate of freshlyharvested TILs. In some embodiments, the basal respiration rate is fromabout 90% to about 99% of the basal respiration rate of freshlyharvested TILs. In some embodiments, the basal respiration rate is fromabout 95% to about 99% of the basal respiration rate of freshlyharvested TILs. In some embodiments, the second expansion or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 11, including TILs referred to as reREP TILs) have a basalrespiration rate that is not statistically significantly different thanthe basal respiration rate of freshly harvested TILs.

In general, second expansion TILs or additional second expansion TILs,such as those in Step D (including, for example, TILs referred to asreREP which have undergone an additional second expansion) TILs have aspare respiratory capacity that is at least is at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, or at least 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the spare respiratory capacity is from about50% to about 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the spare respiratory capacity is from about50% to about 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the spare respiratory capacity is from about60% to about 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the spare respiratory capacity is from about70% to about 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the spare respiratory capacity is from about80% to about 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the spare respiratory capacity is from about90% to about 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the spare respiratory capacity is from about95% to about 99% of the basal respiration rate of freshly harvestedTILs. In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 11, including TILs referred to as reREP TILs) have a sparerespiratory capacity that is not statistically significantly differentthan the basal respiration rate of freshly harvested TILs.

In general, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 11,including TILs referred to as reREP TILs) have a spare respiratorycapacity that is at least is at least 50%, at least 55%, at least 60%,at least 65%, at least 70% at least 75%, at least 80% at least 85% atleast 90% at least 95%, at least 97%, at least 98%, or at least 99% ofthe basal respiration rate of freshly harvested TILs. In someembodiments, the metabolic assay measured is glycolytic reserve. In someembodiments, the metabolic assay is glycolytic reserve. In someembodiments, the metabolic assay is spare respiratory capacity. Tomeasure cellular (respiratory) metabolism cells were treated withinhibitors of mitochondrial respiration and glycolysis to determine ametabolic profile for the TIL consisting of the following measures:baseline oxidative phosphorylation (as measured by OCR), sparerespiratory capacity, baseline glycolytic activity (as measured byECAR), and glycolytic reserve. Metabolic profiles were performed usingthe Seahorse Combination Mitochondrial/Glycolysis Stress Test Assay(including the kit commercially available from Agilent®), which allowsfor determining a cells' capacity to perform glycolysis upon blockage ofmitochondrial ATP production. In some embodiments, cells are starved ofglucose, then glucose is injected, followed by a stress agent. In someembodiments, the stress agent is selected from the group consisting ofoligomycin, FCCP, rotenone, antimycin A and/or 2-deoxyglucose (2-DG), aswell as combinations thereof. In some embodiments, oligomycin is addedat 10 mM. In some embodiments, FCCP is added at 10 mM. In someembodiments, rotenone is added at 2.5 mM. In some embodiments, antimycinA is added at 2.5 mM. In some embodiments, 2-deoxyglucose (2-DG) isadded at 500 mM. In some embodiments, glycolytic capacity, glycolyticreserve, and/or non-glycolytic acidification are measured. In general,TILs have a glycolytic reserve that is at least 50%, at least 55%, atleast 60%, at least 65%, at least 70% at least 75%, at least 80% atleast 85% at least 90% at least 95%, at least 97%, at least 98%, or atleast 99% of the basal respiration rate of freshly harvested TILs. Insome embodiments, the glycolytic reserve is from about 50% to about 99%of the basal respiration rate of freshly harvested TILs. In someembodiments, the glycolytic reserve is from about 60% to about 99% ofthe basal respiration rate of freshly harvested TILs. In someembodiments, the glycolytic reserve is from about 70% to about 99% ofthe basal respiration rate of freshly harvested TILs. In someembodiments, the glycolytic reserve is from about 80% to about 99% ofthe basal respiration rate of freshly harvested TILs. In someembodiments, the glycolytic reserve is from about 90% to about 99% ofthe basal respiration rate of freshly harvested TILs. In someembodiments, the glycolytic reserve is from about 95% to about 99% ofthe basal respiration rate of freshly harvested TILs.

In some embodiments, the metabolic assay is basal glycolysis. In someembodiments second expansion TILs or additional second expansion TILs,such as those in Step D (including, for example, TILs referred to asreREP which have undergone an additional second expansion) have anincrease in basal glycolysis of at least two-fold, at least three-fold,at least four-fold, at least five-fold, at least six-fold, at least7-fold, at least eight-fold, at least nine-fold, or at least ten-fold.In some embodiments, the second expansion TILs or additional secondexpansion, such as those in Step D (including TILs referred to as reREPTILs) have an increase in basal glycolysis of about two-fold to aboutten-fold. In some embodiments, the second expansion TILs or additionalsecond expansion, such as those in Step D (including TILs referred to asreREP TILs) have an increase in basal glycolysis of about two-fold toabout eight-fold. In some embodiments, the second expansion TILs oradditional second expansion, such as those in Step D (including TILsreferred to as reREP TILs) have an increase in basal glycolysis of aboutthree-fold to about seven-fold. In some embodiments, the secondexpansion TILs or additional second expansion, such as those in Step D(including TILs referred to as reREP TILs) have an increase in basalglycolysis of about two-fold to about four-fold. In some embodiments,the second expansion TILs or additional second expansion, such as thosein Step D (including TILs referred to as reREP TILs) have an increase inbasal glycolysis of about two-fold to about three-fold.

In general, second expansion TILs or additional second expansion, suchas those in Step D (including, for example, TILs referred to as reREPwhich have undergone an additional second expansion) TILs have aglycolytic reserve that is at least 50%, at least 55%, at least 60%, atleast 65%, at least 70% at least 75%, at least 80% at least 85% at least90% at least 95%, at least 97%, at least 98%, or at least 99% of thebasal respiration rate of freshly harvested TILs. In some embodiments,the glycolytic reserve is from about 50% to about 99% of the basalrespiration rate of freshly harvested TILs. In some embodiments, theglycolytic reserve is from about 60% to about 99% of the basalrespiration rate of freshly harvested TILs. In some embodiments, theglycolytic reserve is from about 70% to about 99% of the basalrespiration rate of freshly harvested TILs. In some embodiments, theglycolytic reserve is from about 80% to about 99% of the basalrespiration rate of freshly harvested TILs. In some embodiments, theglycolytic reserve is from about 90% to about 99% of the basalrespiration rate of freshly harvested TILs. In some embodiments, theglycolytic reserve is from about 95% to about 99% of the basalrespiration rate of freshly harvested TILs. In some embodiments, thesecond expansion TILs or second additional expansion TILs (such as, forexample, those described in Step D of FIG. 11, including TILs referredto as reREP TILs) have a spare respiratory capacity that is notstatistically significantly different than the basal respiration rate offreshly harvested TILs.

Granzyme B Production: Granzyme B is another measure of the ability ofTIL to kill target cells. Media supernatants restimulated as describedabove using antibodies to CD3, CD28, and CD137/4-1BB were also evaluatedfor their levels of Granzyme B using the Human Granzyme B DuoSet ELISAKit (R & D Systems, Minneapolis, Minn.) according to the manufacturer'sinstructions. In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 11, including TILs referred to as reREP TILs) have increasedGranzyme B production. In some embodiments, the second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 11, including TILs referred to as reREP TILs) haveincreased cytotoxic activity.

In some embodiments, the present methods include an assay for assessingTIL viability, using the methods as described above. In someembodiments, the TILs are expanded as discussed above, including forexample as provided in FIG. 11. In some embodiments, the TILs arecryopreserved prior to being assessed for viability. In someembodiments, the viability assessment includes thawing the TILs prior toperforming a first expansion, a second expansion, and an additionalsecond expansion. In some embodiments, the present methods provide anassay for assessing cell proliferation, cell toxicity, cell death,and/or other terms related to viability of the TIL population. Viabilitycan be measured by any of the TIL metabolic assays described above aswell as any methods know for assessing cell viability that are known inthe art. In some embodiments, the present methods provide as assay forassessment of cell proliferation, cell toxicity, cell death, and/orother terms related to viability of the TILs expanded using the methodsdescribed herein, including those exemplified in FIG. 11.

The present invention also provides assay methods for determining TILviability. The present disclosure provides methods for assaying TILs forviability by expanding tumor infiltrating lymphocytes (TILs) into alarger population of TILs comprising:

-   -   (i) obtaining a first population of TILs which has been        previously expanded;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold or 100-fold greater in number than the        second population of TILs, and wherein the second expansion is        performed for at least 14 days in order to obtain the third        population of TILs, wherein the third population of TILs        comprises an increased subpopulation of effector T cells and/or        central memory T cells relative to the second population of        TILs, and wherein the third population is further assayed for        viability.

In some embodiments, the method further comprises:

-   -   (iv) performing an additional second expansion by supplementing        the cell culture medium of the third population of TILs with        additional IL-2, additional OKT-3, and additional APCs, wherein        the additional second expansion is performed for at least 14        days to obtain a larger population of TILs than obtained in step        (iii), wherein the larger population of TILs comprises an        increased subpopulation of effector T cells and/or central        memory T cells relative to the third population of TILs, and        wherein the third population is further assayed for viability.

In some embodiments, prior to step (i), the cells are cryopreserved.

In some embodiments, the cells are thawed prior to performing step (i).

In some embodiments, step (iv) is repeated one to four times in order toobtain sufficient TILs for analysis.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 40 days to about 50 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 48 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 45 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin about 44 days.

In some embodiments, the cells from steps (iii) or (iv) express CD4,CD8, and TCR αβ at levels similar to freshly harvested cells.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 17 in step (iii).

In some embodiments, the effector T cells and/or central memory T cellsin the larger population of TILs in step (iv) exhibit one or morecharacteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T cells, and/orcentral memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a high-affinity T cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a chimeric antigen receptor (CAR)comprising a single chain variable fragment antibody fused with at leastone endodomain of a T-cell signaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability.

In some embodiments, the TILs are assayed for viability aftercryopreservation.

In some embodiments, the TILs are assayed for viability aftercryopreservation and after step (iv).

According to the present disclosure, a method for assaying TILs forviability and/or further use in administration to a subject. In someembodiments, the method for assay tumor infiltrating lymphocytes (TILs)comprises:

-   -   (i) obtaining a first population of TILs;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs;    -   (iv) harvesting, washing, and cryopreserving the third        population of TILs;    -   (v) storing the cryopreserved TILs at a cryogenic temperature;    -   (vi) thawing the third population of TILs to provide a thawed        third population of TILs; and    -   (vii) performing an additional second expansion of a portion of        the thawed third population of TILs by supplementing the cell        culture medium of the third population with IL-2, OKT-3, and        APCs for a reREP period of at least 3 days, wherein the third        expansion is performed to obtain a fourth population of TILs,        wherein the number of TILs in the fourth population of TILs is        compared to the number of TILs in the third population of TILs        to obtain a ratio;    -   (viii) determining based on the ratio in step (vii) whether the        thawed population of TILs is suitable for administration to a        patient;    -   (ix) administering a therapeutically effective dosage of the        thawed third population of TILs to the patient when the ratio of        the number of TILs in the fourth population of TILs to the        number of TILs in the third population of TILs is determined to        be greater than 5:1 in step (viii).

In some embodiments, the reREP period is performed until the ratio ofthe number of TILs in the fourth population of TILs to the number ofTILs in the third population of TILs is greater than 50:1.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, steps (i) through (vii) are performed within aperiod of about 40 days to about 50 days. In some embodiments, steps (i)through (vii) are performed within a period of about 42 days to about 48days. In some embodiments, steps (i) through (vii) are performed withina period of about 42 days to about 45 days. In some embodiments, steps(i) through (vii) are performed within about 44 days.

In some embodiments, the cells from steps (iii) or (vii) express CD4,CD8, and TCR αβ at levels similar to freshly harvested cells. In someembodiments the cells are TILs.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are added tothe cell culture on any of days 9 through 17 in step (iii).

In some embodiments, the effector T cells and/or central memory T cellsin the larger population of TILs in steps (iii) or (vii) exhibit one ormore characteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T cells, and/orcentral memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding ahigh-affinity T cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding achimeric antigen receptor (CAR) comprising a single chain variablefragment antibody fused with at least one endodomain of a T-cellsignaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability after step(vii).

The present disclosure also provides further methods for assaying TILs.In some embodiments, the disclosure provides a method for assaying TILscomprising:

-   -   (i) obtaining a portion of a first population of cryopreserved        TILs;    -   (ii) thawing the portion of the first population of        cryopreserved TILs;    -   (iii) performing a first expansion by culturing the portion of        the first population of TILs in a cell culture medium comprising        IL-2, OKT-3, and antigen presenting cells (APCs) for a reREP        period of at least 3 days, to produce a second population of        TILs, wherein the portion from the first population of TILs is        compared to the second population of TILs to obtain a ratio of        the number of TILs, wherein the ratio of the number of TILs in        the second population of TILs to the number of TILs in the        portion of the first population of TILs is greater than 5:1;    -   (iv) determining based on the ratio in step (iii) whether the        first population of TILs is suitable for use in therapeutic        administration to a patient;    -   (v) determining the first population of TILs is suitable for use        in therapeutic administration when the ratio of the number of        TILs in the second population of TILs to the number of TILs in        the first population of TILs is determined to be greater than        5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the secondpopulation of TILs to the number of TILs in the portion of the firstpopulation of TILs is greater than 50:1.

In some embodiments, the method further comprises performing expansionof the entire first population of cryopreserved TILs from step (i)according to the methods as described in any of the embodiments providedherein.

In some embodiments, the method further comprises administering theentire first population of cryopreserved TILs from step (i) to thepatient.

In some embodiments, the cryopreserved TILs are thawed and a secondexpansion performed to determine if the cells expand sufficiently. Ifthe cells expand to a ratio of at least 5:1, the TILs are sufficientlyviably for administration to the patient. If the cells expand to a ratioof at least 10:1, the TILs are sufficiently viably for administration tothe patient. If the cells expand to a ratio of at least 15:1, the TILsare sufficiently viably for administration to the patient. If the cellsexpand to a ratio of at least 20:1, the TILs are sufficiently viably foradministration to the patient. If the cells expand to a ratio of atleast 25:1, the TILs are sufficiently viably for administration to thepatient. If the cells expand to a ratio of at least 30:1, the TILs aresufficiently viably for administration to the patient. If the cellsexpand to a ratio of at least 35:1, the TILs are sufficiently viably foradministration to the patient. If the cells expand to a ratio of atleast 40:1, the TILs are sufficiently viably for administration to thepatient. If the cells expand to a ratio of at least 45:1, the TILs aresufficiently viably for administration to the patient. If the cellsexpand to a ratio of at least 5:1, the TILs are sufficiently viably foradministration to the patient.

The present disclosure also provides further methods for assaying TILs.In some embodiments, the disclosure provides a method for assaying TILscomprising:

-   -   (i) obtaining a portion of a first population of cryopreserved        TILs;    -   (ii) thawing the portion of the first population of        cryopreserved TILs;    -   (iii) performing a first expansion by culturing the portion of        the first population of TILs in a cell culture medium comprising        IL-2, OKT-3, and antigen presenting cells (APCs) for a reREP        period of at least 3 days, to produce a second population of        TILs, wherein the portion from the first population of TILs is        compared to the second population of TILs to obtain a ratio of        the number of TILs, wherein the ratio of the number of TILs in        the second population of TILs to the number of TILs in the        portion of the first population of TILs is greater than 5:1;    -   (iv) determining based on the ratio in step (iii) whether the        first population of TILs is suitable for use in therapeutic        administration to a patient; and    -   (v) therapeutically administering the remainder of the first        population of TILs to the patient when the ratio of the number        of TILs in the second population of TILs to the number of TILs        in the first population of TILs is determined to be greater than        5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the secondpopulation of TILs to the number of TILs in the portion of the firstpopulation of TILs is greater than 50:1.

In some embodiments, the method further comprises performing expansionof the entire first population of cryopreserved TILs from step (i)according to the methods of any of the preceding claims.

In some embodiments, the method further comprises administering theentire first population of cryopreserved TILs from step (i) to thepatient.

In some embodiments, the method further comprised the step of assessingthe metabolic health of the second population of TILs.

In some embodiments, the method further comprises the step of assessingthe phenotype of the second population of TILs.

In some embodiments, the antigen presenting cells are allogeneicperipheral blood mononuclear cells.

L. Methods of Treating Patients

Methods of treatment begin with the initial TIL collection and cultureof TILs. Such methods have been both described in the art by, forexample, Jin et al. Immunotherapy, 2012, 35(3):283-292), incorporated byreference herein in its entirety. As well as described throughout theExamples section below.

The present invention provides novel methods for TIL generation thathave not been previously described, e.g., TILs produced according toSteps A through F. The expanded TILs produced according to Steps Athrough F above or as otherwise produced as described herein findparticular use in the treatment of patients with cancer. General methodsof using TILs for the treatment of cancer have been described in Goff,et al., J. Clinical Oncology, 2016, 34(20):2389-239, as well as thesupplemental content; incorporated by reference herein in its entirety.)Similarly, the TILs produced according to the present invention can alsobe used for the treatment of cancer. In some embodiments, TIL were grownfrom resected deposits of metastatic melanoma as previously described(see, Dudley, et al., J Immunother 2003, 26:332-342; incorporated byreference herein in its entirety). Fresh tumor can be dissected understerile conditions. A representative sample can be collected for formalpathologic analysis. Single fragments of 2 mm³ to 3 mm³. In someembodiments, 5, 10, 15, 20, 25 or 30 samples per patient are obtained.In some embodiments, 20, 25, or 30 samples per patient are obtained. Insome embodiments, 20, 22, 24, 26, or 28 samples per patient areobtained. In some embodiments, 24 samples per patient are obtained.Samples can be placed in individual wells of a 24-well plate, maintainedin growth media with high-dose IL-2 (6,000 IU/mL), and monitored fordestruction of tumor and/or proliferation of TIL. Any tumor with viablecells remaining after processing can be enzymatically digested into asingle cell suspension and cryopreserved, as described herein.

In some embodiments, expanded TILs can be sampled for phenotype analysis(CD3, CD4, CD8, and CD56) and tested against autologous tumor whenavailable. TILs can be considered reactive if overnight co-cultureyielded interferon-gamma (IFN-γ) levels >200 pg/mL and twice background.(Goff, et al., J Immunother., 2010, 33:840-847; incorporated byreference herein in its entirety). In some embodiments, cultures withevidence of autologous reactivity or sufficient growth patterns can beselected for a second expansion (for example, a second expansion asprovided in according to Step D of FIG. 11), including second expansionsthat are sometimes referred to as rapid expansion (REP). In someembodiments, expanded TILs with high autologous reactivity (for example,high proliferation during a second expansion), are selected for anadditional second expansion. In some embodiments, TILs with highautologous reactivity (for example, high proliferation during secondexpansion as provided in Step D of FIG. 11), are selected for anadditional second expansion according to Step D of FIG. 11.

In some embodiments, the patient is not moved directly to ACT (adoptivecell transfer), for example, in some embodiments, after tumor harvestingand/or a first expansion, the cells are not utilized immediately. Insuch embodiments, TILs can be cryopreserved and thawed 2 days before thesecond expansion step (for example, in some embodiments, 2 days before astep referred to as a REP step). In such embodiments, TILs can becryopreserved and thawed 2 days before the second expansion step (forexample, in some embodiments, 2 days before a Step D as provided in FIG.11). As described in various embodiments throughout the presentapplication, the second expansion (including processes referred to asREP) used OKT3 (anti-CD3) antibody (Miltenyi Biotech, San Diego, Calif.)and IL-2 (3,000 IU/mL; Prometheus, San Diego, Calif.) in the presence ofirradiated feeder cells, autologous when possible, at a 100:1 ratio(see, Dudley, et al., J Immunother., 2003, 26:332-342; incorporated byreference herein in its entirety). In some embodiments, the TILs can becryopreserved and thawed 5 days before the second expansion step. Insome embodiments, the TILs can be cryopreserved and thawed 4 days beforethe second expansion step. In some embodiments, the TILs can becryopreserved and thawed 3 days before the second expansion step. Insome embodiments, the TILs can be cryopreserved and thawed 2 days beforethe second expansion step. In some embodiments, the TILs can becryopreserved and thawed 1 day before the second expansion step. In someembodiments, the TILs can be cryopreserved and thawed immediately beforethe second expansion step.

Cell phenotypes of cryopreserved samples of infusion bag TIL can beanalyzed by flow cytometry (FlowJo) for surface markers CD3, CD4, CD8,CCR7, and CD45RA (BD BioSciences), as well as by any of the methodsdescribed herein. Serum cytokines were measured by using standardenzyme-linked immunosorbent assay techniques. A rise in serum IFN-g wasdefined as >100 pg/mL and greater than 4 3 baseline levels.

1. Optional Lymphodepletion Preconditioning of Patients

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (‘cytokine sinks’). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 11, including TILs referred to as reREP TILs) of theinvention.

In general, lymphodepletion is done using fludarabine and/orcyclophosphamide (the active form being referred to as mafosfamide) andcombinations thereof. Such methods are described in Gassner et al.(Cancer Immunol Immunother. 2011, 60(1):75-85, Muranski, et al., NatClin Pract Oncol., 2006 3(12):668-681, Dudley, et al., J Clin Oncol2008, 26:5233-5239, and Dudley, et al., J Clin Oncol. 2005,23(10):2346-2357, all of which are incorporated by reference herein intheir entireties.

In some embodiments, the fludarabine is at a concentration of 0.5μg/ml-10 μg/ml fludarabine (Sigma-Aldrich, MO, USA). In someembodiments, the fludarabine is at a concentration of 1 μg/mlfludarabine (Sigma-Aldrich, MO, USA). In some embodiments, thefludarabine treatment is for 1 day, 2 days, 3 days, 4 days, 5 days, 6days, or 7 days or more. In some embodiments, the fludarabine isadministered at a dosage of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. Insome embodiments, the fludarabine treatment is for 2-7 days at 35mg/kg/day. In some embodiments, the fludarabine treatment is for 4-5days at 35 mg/kg/day. In some embodiments, the fludarabine treatment isfor 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form ofcyclophosphamide, is at a concentration of 0.5 μg/ml-10 μg/ml. In someembodiments, the mafosfamide, the active form of cyclophosphamide, is ata concentration of 1 μg/ml. In some embodiments, the cyclophosphamidetreatment is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7days or more. In some embodiments, the cyclophosphamide is administeredat a dosage of 100 mg/m²/day, 150 mg/m²/day, 175 mg/m²/day 200mg/m²/day, 225 mg/m²/day, 250 mg/m²/day, 275 mg/m²/day, or 300mg/m²/day. In some embodiments, the cyclophosphamide is administeredintravenously (i.e., i.v.) In some embodiments, the cyclophosphamidetreatment is for 2-7 days at 35 mg/kg/day. In some embodiments, thecyclophosphamide treatment is for 4-5 days at 250 mg/m²/day i.v. In someembodiments, the cyclophosphamide treatment is for 4 days at 250mg/m²/day i.v.

In some embodiments, the fludarabine and the cyclophosphamide areadministered together to a patient. In some embodiments, fludarabine isadministered at 25 mg/m²/day i.v. and cyclophosphamide is administeredat 250 mg/m²/day i.v. over 4 days.

This protocol includes administration of fludarabine (25 mg/m²/day i.v.)and cyclophosphamide (250 mg/m²/day i.v.) over 4 days.

2. Exemplary Treatment Embodiments

In some embodiments, the present disclosure provides a method oftreating a cancer with a population of tumor infiltrating lymphocytes(TILs) comprising the steps of (a) obtaining a first population of TILsfrom a tumor resected from a patient; (b) performing an initialexpansion of the first population of TILs in a first cell culture mediumto obtain a second population of TILs, wherein the second population ofTILs is at least 5-fold greater in number than the first population ofTILs, and wherein the first cell culture medium comprises IL-2; (c)performing a rapid expansion of the second population of TILs using apopulation of myeloid artificial antigen presenting cells (myeloidaAPCs) in a second cell culture medium to obtain a third population ofTILs, wherein the third population of TILs is at least 50-fold greaterin number than the second population of TILs after 7 days from the startof the rapid expansion; and wherein the second cell culture mediumcomprises IL-2 and OKT-3; (d) administering a therapeutically effectiveportion of the third population of TILs to a patient with the cancer. Insome embodiments, the IL-2 is present at an initial concentration ofabout 3000 IU/mL and OKT-3 antibody is present at an initialconcentration of about 30 ng/mL in the second cell culture medium. Insome embodiments, first expansion is performed over a period not greaterthan 14 days. In some embodiments, the first expansion is performedusing a gas permeable container. In some embodiments, the secondexpansion is performed using a gas permeable container. In someembodiments, the ratio of the second population of TILs to thepopulation of aAPCs in the rapid expansion is between 1 to 80 and 1 to400. In some embodiments, the ratio of the second population of TILs tothe population of aAPCs in the rapid expansion is about 1 to 300. Insome embodiments, the cancer for treatment is selected from the groupconsisting of melanoma, ovarian cancer, cervical cancer, non-small-celllung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancercaused by human papilloma virus, head and neck cancer, renal cancer, andrenal cell carcinoma. In some embodiments, the cancer for treatment isselected from the group consisting of melanoma, ovarian cancer, andcervical cancer. In some embodiments, the cancer for treatment ismelanoma. In some embodiments, the cancer for treatment is ovariancancer. In some embodiments, the cancer for treatment is cervicalcancer. In some embodiments, the method of treating cancer furthercomprises the step of treating the patient with a non-myeloablativelymphodepletion regimen prior to administering the third population ofTILs to the patient. In some embodiments, the non-myeloablativelymphodepletion regimen comprises the steps of administration ofcyclophosphamide at a dose of 60 mg/m2/day for two days followed byadministration of fludarabine at a dose of 25 mg/m2/day for five days.In some embodiments, the high dose IL-2 regimen comprises 600,000 or720,000 IU/kg of aldesleukin, or a biosimilar or variant thereof,administered as a 15-minute bolus intravenous infusion every eight hoursuntil tolerance.

3. Methods of Co-Administration

In some embodiments, the TILs produced as described herein in Steps Athrough F can be administered in combination with one or more immunecheckpoint regulators, such as the antibodies described below. Forexample, antibodies that target PD-1 and which can be co-administeredwith the TILs of the present invention include, e.g., but are notlimited to nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®),pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®),humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonalanti-PD-1 antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAbCT-011, Medivation), anti-PD-1 monoclonal Antibody BGB-A317 (BeiGene),and/or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonalantibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106(Bristol-Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001(Novartis). In some embodiments, the PD-1 antibody is from clone:RMP1-14 (rat IgG)—BioXcell cat #BP0146. Other suitable antibodiessuitable for use in co-administration methods with TILs producedaccording to Steps A through F as described herein are anti-PD-1antibodies disclosed in U.S. Pat. No. 8,008,449, herein incorporated byreference. In some embodiments, the antibody or antigen-binding portionthereof binds specifically to PD-L1 and inhibits its interaction withPD-1, thereby increasing immune activity. Any antibodies known in theart which bind to PD-L1 and disrupt the interaction between the PD-1 andPD-L1, and stimulates an anti-tumor immune response, are suitable foruse in co-administration methods with TILs produced according to Steps Athrough F as described herein. For example, antibodies that target PD-L1and are in clinical trials, include BMS-936559 (Bristol-Myers Squibb)and MPDL3280A (Genentech). Other suitable antibodies that target PD-L1are disclosed in U.S. Pat. No. 7,943,743, herein incorporated byreference. It will be understood by one of ordinary skill that anyantibody which binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1interaction, and stimulates an anti-tumor immune response, are suitablefor use in co-administration methods with TILs produced according toSteps A through F as described herein. In some embodiments, the subjectadministered the combination of TILs produced according to Steps Athrough F is co-administered with a and anti-PD-1 antibody when thepatient has a cancer type that is refractory to administration of theanti-PD-1 antibody alone. In some embodiments, the patient isadministered TILs in combination with and anti-PD-1 when the patient hasrefactory melanoma. In some embodiments, the patient is administeredTILs in combination with and anti-PD-1 when the patient has non-smallcell lung carcinoma (NSCLC).

4. Adoptive Cell Transfer

Adoptive cell transfer (ACT) is a very effective form of immunotherapyand involves the transfer of immune cells with antitumor activity intocancer patients. ACT is a treatment approach that involves theidentification, in vitro, of lymphocytes with antitumor activity, the invitro expansion of these cells to large numbers and their infusion intothe cancer-bearing host. Lymphocytes used for adoptive transfer can bederived from the stroma of resected tumors (tumor infiltratinglymphocytes or TILs). TILs for ACT can be prepared as described herein.In some embodiments, the TILs are prepared, for example, according to amethod as described in FIG. 11. They can also be derived or from bloodif they are genetically engineered to express antitumor T-cell receptors(TCRs) or chimeric antigen receptors (CARs), enriched with mixedlymphocyte tumor cell cultures (MLTCs), or cloned using autologousantigen presenting cells and tumor derived peptides. ACT in which thelymphocytes originate from the cancer-bearing host to be infused istermed autologous ACT. U.S. Publication No. 2011/0052530 relates to amethod for performing adoptive cell therapy to promote cancerregression, primarily for treatment of patients suffering frommetastatic melanoma, which is incorporated by reference in its entiretyfor these methods.

In some embodiments, TILs can be administered as described herein. Insome embodiments, TILs can be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs and/or cytotoxic lymphocytes may be administered inmultiple doses. Dosing may be once, twice, three times, four times, fivetimes, six times, or more than six times per year. Dosing may be once amonth, once every two weeks, once a week, or once every other day.Administration of TILs and/or cytotoxic lymphocytes may continue as longas necessary.

I. Exemplary Embodiments

In an embodiment, the invention provides a method for expanding tumorinfiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the first cell culture medium        comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs, wherein the third population of TILs is at least 100-fold        greater in number than the second population of TILs; and        wherein the second cell culture medium comprises IL-2, OKT-3,        and peripheral blood mononuclear cells (PBMCs), wherein the        rapid expansion is performed for at least 14 days;    -   (d) removing the cells from the second cell culture medium and        optionally cryopreserving the cells in a storage medium to        obtain a third population of cells;    -   (e) optionally thawing the third population of cells; and    -   (f) performing a second rapid expansion of the third population        of TILs in a third cell culture medium, wherein the third cell        culture medium comprises IL-2, OKT-3, and peripheral blood        mononuclear cells (PBMCs), wherein the second rapid expansion is        performed for at least 14 days, to obtain a fourth population of        TILs, wherein the fourth population of cells exhibits an        increased subpopulation of effector T cells and/or central        memory T cells relative to the second population of TILs; and    -   g) optionally, repeating step f) one or more times.

In an embodiment, the invention provides that said restimulated cellsexpress CD4, CD8 and TCR αβ at levels similar to freshly harvestedcells.

In an embodiment, the invention provides that said reREP mediumcomprises peripheral blood mononuclear cells (PBMCs).

In an embodiment, the invention provides that said PBMCs are added tothe TILs on any of days 9 through 17. In some embodiments, the inventionprovides that said PBMCs are added to the TILs on days 9, 10, 11, 12,13, 14, 15, 16, and/or 17.

In an embodiment, the invention provides that said reREP mediumcomprises aAPCs.

In an embodiment, the invention provides that the cryopreserved TILswere transduced with an expression vector comprising a nucleic acidencoding a high-affinity T cell receptor.

In an embodiment, the invention provides that the cryopreserved TILswere transduced with an expression vector comprising a nucleic acidencoding a chimeric antigen receptor (CAR) comprising an immunoglobulinlight chain fused with an endodomain of a T-cell signaling molecule.

In an embodiment, the invention provides that restimulated TILs areinfused into a patient.

In an embodiment, the invention provides that step d) further comprisesremoving the cells from the second cell culture medium.

In an embodiment, the invention provides that step f) is repeated asufficient number of times in order to obtain sufficient TILs for atherapeutic dosage of said TILs.

In an embodiment, the invention provides a population of restimulatedTILs made according to the methods described above and herein.

In an embodiment, the invention provides a population of restimulatedTILs made according to the method of claim 1 wherein said restimulatedTILs have at least a two-fold increase in basal glycolysis as comparedto said thawed cryopreserved TILs.

In an embodiment, the invention provides a method for assessing themetabolic activity of a TIL cell population comprising measuring thebasal glycolysis of said cells.

In an embodiment, the invention provides a method for assessing themetabolic activity of a TIL cell population comprising measuring thebasal respiration of said cells.

In an embodiment, the invention provides a method for assessing themetabolic activity of a TIL cell population comprising measuring thespare respiratory capacity (SRC) of said cells.

In an embodiment, the invention provides a method for assessing themetabolic activity of a TIL cell population comprising measuring theglycolytic reserve of said cells.

In an embodiment, the invention provides a method of treating cancer ina patient with a population of tumor infiltrating lymphocytes (TILs)comprising the steps of:

-   -   a) obtaining a primary TIL population from said patient;    -   b) rapidly expanding said primary TIL population to form an        expanded TIL population;    -   c) cryopreserving said expanded population to form a        cryopreserved TIL population;    -   d) thawing said cryopreserved TIL population;    -   e) culturing said cryopreserved TIL population in media        comprising IL-2 and anti-CD3 antibody to form a reREP TIL        population; and    -   f) administering a therapeutically effective amount of reREP TIL        cells to said patient.

In an embodiment, the invention provides a method for expanding tumorinfiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the first cell culture medium        comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs, wherein the third population of TILs is at least 100-fold        greater in number than the second population of TILs; and        wherein the second cell culture medium comprises IL-2, OKT-3,        and peripheral blood mononuclear cells (PBMCs), wherein the        rapid expansion is performed for at least 14 days;    -   (d) removing the cells from the second cell culture medium and        optionally cryopreserving the cells in a storage medium to        obtain a third population of cells;    -   (e) optionally thawing the third population of cells;    -   (f) performing a second rapid expansion of the third population        of TILs in a third cell culture medium, wherein the third cell        culture medium comprises IL-2, OKT-3, and peripheral blood        mononuclear cells (PBMCs), wherein the second rapid expansion is        performed for at least 14 days, to obtain a fourth population of        TILs, wherein the fourth population of cells exhibits an        increased subpopulation of effector T cells and/or central        memory T cells relative to the second population of TILs; and    -   (g) administering a therapeutically effective amount of reREP        TIL cells to said patient.

In an embodiment, the invention provides that step d) further comprisesremoving the cells from the second cell culture medium.

In an embodiment, the invention provides that step f) is repeated asufficient number of times in order to obtain sufficient TILs for atherapeutic dosage of said TILs.

EXAMPLES Example 1: Restimulation Protocol

As discussed herein, a restimulation protocol and assay were developedutilizing fresh antigen restimulation following harvest or thaw of TILsgrown in a REP.

The purpose of this example was to test the proliferation/expansion ofpost REP Tumor Infiltrating Lymphocytes in a Re-stimulation assay. PostREP TIL (post Step D TIL according to FIG. 11) were be restimulated withallogeneic PBMC feeder cells, anti-CD3 (clone OKT3) antibody, andinterleukin-2 (IL-2). Viable cells were counted on Day 7 and recorded.

The post REP TIL (post Step D TIL according to FIG. 11) were infusedinto the patients who were previously lymphodepleted to facilitate TILsurvival and expansion in vivo. Once the TIL were re-infused into thepatient, they encountered antigen, resulting in the activation of theTIL, but the TIL were ultimately short-lived. Re-stimulation of the TILthrough antigen contact together with exposure to IL-2 during ACT mayresult in TIL proliferation and tumor control or may lead to deletionthrough apoptosis (activation induced cell death) or induction of anon-proliferative (anergic) state due to lack of appropriateco-stimulation. Without being bound by theory, restimulation of post REPTIL (restimulation of, for example post Step D TIL according to FIG. 11)with allogeneic PBMC feeder cells may mimic the in vivo process byproviding antigen stimulation and necessary cytokines for TIL expansion.Post REP TIL (post Step D TIL according to FIG. 11) were activatedthrough membrane receptors on the feeder MNCs that bind to anti-CD3(clone OKT3) antibody and crosslink to TIL in the REP flask, stimulatingthe TIL to expand.

Proliferation/Expansion of Post REP Tumor Infiltrating Lymphocytes in aRe-Stimulation Assay

Post REP (post Step D TIL according to FIG. 11) TIL were restimulatedwith allogeneic PBMC feeder cells, anti-CD3 (clone OKT3) antibody, andinterleukin-2 (IL-2). Viable cells were counted on Day 7 and recorded.

In some embodiments, this procedure can also be applied to test orvalidate the current REP protocol.

TABLE 3 DEFINITIONS AND ABBREVIATIONS Abbreviation Definition μlMicroliter AOPI Acridine Orange Propidium Iodide BSC Biological SafetyCabinet BSL2 Biosafety Level 2 CM1 Complete Medium for TIL, #1 CM2Complete Medium for TIL, #2; 50:50 mixture of CM1 and AIM-V GMP GoodManufacturing Processing Gy Gray IPA Isopropyl alcohol LN2 Liquidnitrogen MNC; PBMC Mononuclear Cells; Peripheral Blood Mononuclear Cellsml Milliliter NA Not applicable NR Not required OKT3 MACS ® GMP CD3 pure(clone OKT3) antibody PPE Personal protective equipment Pre-REP InitialTIL cultures originating from tumor fragments REP Rapid ExpansionProtocol SDBB San Diego Blood Bank TIL Tumor Infiltrating Lymphocyte

TABLE 4 Materials Product Specifications Vendor Catalog # Storage AIM-VGMP Gibco ™/Life 087-0112DK 2-8° C. Technologies Cellometer ViaStain ™NA Nexcelom CS2-0106 2-8° C. AOPI Staining Solution Disposable NANexcelom CP2-001 RT Hemacytometer CM1 Prepared as per NA NA 2-8° C.Example 5 GMP recombinant 6 × 10⁶ IU/ml stock CellGenix 1020-1000 −20°C.  human IL-2 (rhIL-2) solution prepared as per Example 4 MACS ® GMPCD3 pure GMP Miltenyi Biotec 170-076-116 2-8° C. (clone OKT3) antibody50 ml conical tubes sterile Any in use RT transfer pipets sterile Any inuse RT 500 ml filter system sterile EMD/Millipore SCGPU05RE or RT orequivalent equivalent 24-well tissue culture sterile Greiner or 662160or RT plates equivalent equivalent 5 ml, 10 ml serological sterile Anyin use RT pipets Pipet tips sterile Any in use RT

TABLE 5 SPECIMENS Ref Specimen Specification Origin number StorageCryopreserved and Stored SDBB NA NA Gamma-irradiated infreezer MNCFeeder lots Post-REP TIL cells Fresh or Iovance NA NA Frozen inBiotechnologies freezer

The post REP (post Step D TIL according to FIG. 11) TIL were infusedinto the patients who were prior lymphodepleted to facilitate TILsurvival and expansion in vivo. Once the TIL were re-infused into thepatient, they encountered antigen, resulting in the activation of theTIL, but the TIL were ultimately short-lived. Re-stimulation of the TILthrough antigen contact together with exposure to IL-2 during ACT mayresult in TIL proliferation and tumor control or may lead to deletionthrough apoptosis (activation induced cell death) or induction of anon-proliferative (anergic) state due to lack of appropriateco-stimulation. Our hypothesis was that restimulation of post REP TILwith allogeneic PBMC feeder cells mimicked the in vivo process byproviding antigen stimulation and necessary cytokines for TIL expansion.Post REP TIL were activated through membrane receptors on the feederMNCs that bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL inthe REP flask, stimulating the TIL to expand.

Procedure

Either fresh post-REP (post Step D TIL according to FIG. 11) or frozenpost-REP (post Step D TIL according to FIG. 11) TIL that was thawed, waswashed once in CM1 media. The Re-REP (repeat of Step D according to FIG.11) was set up in a 24 well tissue culture plate with 2×10⁶ MNC feedercells, 30 ng/ml OKT3, 1×10⁴ post-REP TIL plus 3,000 IU/ml rhIL-2 in CM2.The cultures were incubated for seven days in a 5% CO₂, 37° C.humidified incubator at which point viable cell recovery and viabilitywas determined. The fold expansion of TIL was calculated based on theviable cell counts.

ReREP—Day 0

Prepare TIL TILs were obtained from fresh post REP or frozen post REP.TIL cultures were removed from the incubator and transferred to the BSC.Next, 200 μl was removed for a cell count using the Cellometer K2.Counts were recorded.

Prepare Feeder Cells

For this protocol a minimum of 20×10⁶ feeder cells were needed. Each 1ml vial frozen by SDBB had 100×10⁶ viable cells upon freezing. Assuminga 50% recovery upon thaw from LN2 storage, it was recommended to thaw atleast two vials of feeder cells per lot giving an estimated 100×10⁶viable cells for each REP. Before thawing feeder cells, approximately 50ml of CM2 was pre-warmed without rhIL-2 for each feeder lot that wastested. The designated feeder lot vials were removed from LN2 storageand placed on ice. Vials were transferred to the tissue culture room.Vials were thawed in a 37° C. water bath. Vials were transferred to BSCand sprayed or wiped with 70% EtOH or IPA. Using a transfer pipette, thecontents of feeder vials was immediately transferred into 50 mL of warmCM2 in a 50-mL conical tube. 200 μl was removed for cell counting usingthe Cellometer K2. Counts were recorded. Cells were centrifuged at 350×gfor 10 minutes. The supernatant and resuspended cells were aspirated ina desired volume at 2×10⁶ cells/ml in warm CM2 plus 3000 IU/ml rhIL-2.

Prepare CM2+3000 IU/ml Working Solution

A sufficient amount of CM2 was prepared for the conditions needed. Eachwell contained 2 ml of CM2. Each well was supplemented the CM2 with 3000IU/mL of rhIL-2. From the stock of 6×10⁶ IU/mL, 50 μl was needed foreach 100 ml of CM2.

Prepare MACS® GMP CD3 Pure (OKT3) Working Solution

Stock solution of OKT3 (1 mg/ml) was taken out of the 4° C.refrigerator. A final concentration of 30 ng/ml OKT3 was used in theREP. 60 ng of OKT3 were needed for 2 ml of CM2 medium in each 24 well.TIL Feeders, TIL alone and Feeders alone conditions were cultured intriplicates. For each feeder lot tested, 1000 μl of a 1:1000 dilution of1 mg/ml OKT3 for a working concentration of 1 μg/ml (1,000 ng/ml) wasmade. For 9 wells, 1000 μl of a 1:1000 dilution of 1 mg/ml OKT3. 1 μl 1mg/ml OKT3+999 μl of CM2 with 3000 IU/ml IL-2 was made.

Prepare 24 Well Plate and Coculture.

Each ReREP tested required 9 wells of 24 well plate.

Each plate was labeled with Experiment Name, Feeder Lot #, post-REP TILdesignation, date, and operator initials. Each plate was filled withcomponents as listed in Table 8. Each component was added and each wellfilled with a total of 2 ml and place the plates into 37° C. incubator.Plates were mixed carefully 3 times using 1 ml pipette.

TABLE 6 REP set-up in 24 well plate Order of addition to single TIL +Feeders + TIL + Feeders + well of 24 well plate OKT3 OKT3 OKT3 TIL cells(1 × 10⁴/0.5 ml) in 500 μl  500 μl — CM2 + IL-2 PBMC feeder cells (2 ×10⁶/1 1000 μl  — 1000 μl ml) in CM2 + IL-2 OKT3 (1000 ng/ml) in CM2 + 60 μl  60 μl  60 μl IL-2 CM2 + IL-2 440 μl 1440 μl  940 μl Total Volume2000 μl  2000 μl 2000 μlMedia Exchange—Day 5

CM2 was prepared with 3000 IU/ml rhIL-2. 10 ml was needed. 1 ml of themedia was removed from each well and discarded. With a 1 ml pipette, 1ml warm CM2 with 3000 IU/mL rhIL-2 was transferred to each well. Theplates were returned to the incubator.

Harvest—Day 7

Using a 1 ml serological pipet, each well was mixed to break up anyclumps of cells. After thoroughly mixing cell suspension by pipetting,200 μl was removed for cell counting using the Cellometer K2. All theconditions were counted and recorded for TIL+Feeders+OKT3, TIL+OKT3, andFEEDERS+OKT3.

In addition to 24 well ReREP, separate reREP were set up in 4 uprightT25 tissue culture flasks with 1.3×10⁷ MNC feeder cells, 30 ng/ml OKT3,0.65×10⁵ pre-REP TIL plus 3,000 IU/ml rhIL-2 in CM2. Note: Please referto Evaluation of Irradiated Allogeneic Feeder Cells for Rapid ExpansionProtocol of LN-144 (Example 6).

Allocation of cells for functional assays:

TABLE 7 Assay Number of Cells/ Functional Assay Culture Supernatant FlowPhenotyping 10⁶  Potency - P815effLuc-eGFP 40⁶  For restimulation assayto Granzyme-B, IFN- 5⁶ gamma Metabolism 2⁶ TCR Sequencing 1⁶ Storeculture supernatant of TIL + feeders and 1 ml feeders alone forMultiplex ELISAEvaluation/Acceptance Criteria

TABLE 8 Acceptance Criteria Used Test Acceptance criteria TIL expansionAt least a 50-200-fold expansion of Post REP TIL with feeders PBMCFeeders No expansion and at least 20% reduction in cells alone the totalviable number of feeder cellsReference Procedures—Included in Examples Below

TABLE 9 Reference Procedures Name Example Citation Determination of CellCount and Example 2 Viability of TIL Cultures Using the Cellometer K2Cell Counter Preparation of IL-2 stock solution Example 4 (CellGenix) CMMedia Formulation Example 5 Evaluation of Irradiated Allogeneic Example6 Feeder Cells for Rapid Expansion Protocol of LN-144 Extended Phenotypeof Tumor Example 6 infiltrating Lymphocyte after Post REP Validating thepost REP cryofrozen Example 8 and 9 TIL product

Example 2: Determination of Cell Count and Viability of TIL CulturesUsing the Cellometer K2 Cell Counter

This example provides exemplary instructions for how the operation ofthe Cellometer K2 Image Cytometer automatic cell counter was carriedout.

Scope: Determination of the total cell count and viability of cellcultures.

TABLE 10 Definitions μl Microliter AOPI Acridine Orange Propidium IodineBSC Biological Safety Cabinet DPBS Dulbecco's Phosphate Buffered Salineml Milliliter MNC Mononuclear Blood Cells NA Not Applicable PBMCPeripheral Blood Mononuclear Cells PPE Personal Protective EquipmentPre-REP Initial TIL culture before Rapid Expansion Protocol of cultureREP Rapid Expansion Protocol TIL Tumor Infiltrating LymphocytesProcedureCell Suspension PreparationTrypan Blue Preparation

The final Trypan blue concentration was 0.1%. The manufacturerrecommended preparing a stock solution of 0.2%. When using Trypan blueon the Cellometer K2, the stock (0.4%) with PBS was diluted to 0.2%. TheTrypan blue was filtered with a 0.2-0.4 micron filter and aliquoted insmall volumes into labeled, capped tubes. The cell suspension was mixedat 1:1 with 0.2% trypan blue.

AOPI Preparation

When using AOPI on the Cellometer K2, the AOPI solution was obtained.Cell samples were stained at 1:1 with AOPI solution. NOTE: When countinghigh concentration cultures, the cell samples were diluted in cellculture medium prior to the final 1:1 dilution with Trypan Blue or AOPI.The manufacturer's suggested range of counting was used to determine thebest dilution to use.

Cellometer K2 Set-Up

The Cellometer K2 equipment was turned on. The Cellometer ImageCytometer icon was selected on the associated computer monitor. On themain screen of the software, one of the Assays listed in the dropdownbox was selected. When selecting the appropriate Assay, the Cell Typeand Image Mode self-populated. Under “Sample” section, Set User/SampleID was clicked to open another screen to input operator's informationfor specimen. “User ID” was entered. This consisted of the user's threeletter initials. Enter “Sample ID”. The sample ID was derived fromincoming specimen information.

Set Up Dilution Parameters

When no other dilution was made besides the 1:1 mixture, the dilutionfactor was 2. When a dilution was made prior to the final 1:1 mixture,the dilution factor was 2 times of the prior dilution. The dilutionfactor was updated according to the mixture used.

Cell Counting

The plastic backing was removed from both sides of a Cellometer countingchamber slide (SD100) and placed on top of a clean, lint-free wipe.After preparing the cell suspension, a small aliquot of the sample wasremoved and transferred into a well of a multiwell cell culture plate ortube. When diluting the sample, the dilution was performed using cellculture medium. 20 μl of cell suspension was added into a well of themultiwell cell culture plate or tube. 20 μl of 0.2% trypan blue or theAOPI solution was added to the 20 μl of cell suspension and the samplemixed thoroughly. 20 μl of the 1:1 solution was measured and transferredit into one side of the counting chamber. NOTE: Touching the clear areaof the slide was avoided. As needed, the samples were repeated on theother side of the slide. The chamber was inserted into the slot on thefront of the Cellometer. For the AOPI cell counting, “Preview F1” wasselected on the main screen to preview the green fluorescent image (livecell) image. For Trypan blue counting, “Preview Brightfield” wasselected. The focusing wheel was used to bring image into optimal focus.Cells that had a bright center and a clearly-defined edge. “Count” wasselected to begin the counting process. Results were displayed in acounting results pop-up box on the computer screen that showed theresults of the counting process.

Example 3: Cellometer IC2 Image Cytometer Automatic Cell Counter

This Example describes the procedure for operation of the Cellometer K2Image Cytometer automatic cell counter.

1. Definitions

μ; Microliter

AOPI Acridine Orange Propidium Iodine

BSC Biological Safety Cabinet

DPBS Dulbecco's Phosphate Buffered Saline

ml Milliliter

MNC Mononuclear Blood Cells

NA Not Applicable

PBMC Peripheral Blood Mononuclear Cells

PPE Personal Protective Equipment

Pre-REP Initial TIL culture before Rapid Expansion Protocol of culture

REP Rapid Expansion Protocol

TIL Tumor Infiltrating Lymphocytes

7. Procedure

7.1 Cell suspension preparation

-   -   7.1.1 Trypan Blue Preparation    -   The final Trypan blue concentration was 0.1%. The manufacturer        recommended preparing a stock solution of 0.2%.        -   7.1.1.1 When Trypan blue was used on the Cellometer K2, the            stock (0.4%) was diluted with PBS to 0.2%.        -   7.1.1.2 The Trypan blue was filtered with a 0.2-0.4 micron            filter and aliquoted in small volumes into labeled, capped            tubes.        -   7.1.1.3 The cell suspension was mixed at 1:1 with 0.2%            trypan blue.    -   7.1.2 AOPI Preparation        -   7.1.2.1 When AOPI was used on the Cellometer K2, the AOPI            solution was obtained.        -   7.1.2.2 The cell sample was stained at 1:1 with AOPI            solution.    -   NOTE: When high concentration cultures were counted, the cell        samples were diluted in cell culture medium prior to the final        1:1 dilution with Trypan Blue or AOPI.    -   The manufacturer's suggested range of counting was used to        determine the best dilution to use.

7.2 Cellometer K2 Set-Up

-   -   7.2.1 The Cellometer K2 equipment was turned on.    -   7.2.2 The Cellometer Image Cytometer icon was selected on the        associated computer monitor.    -   7.2.3 On the main screen of the software, one of the Assays        listed in the dropdown box was selected.        -   7.2.3.1 When the appropriate Assay was selected, the Cell            Type and Image Mode self-populated.        -   7.2.3.2 Under “Sample” section, Set User/Sample ID was            selected to open another screen to input operator's            information for specimen.            -   7.2.3.2.1 The “User ID” was entered.            -   7.2.3.2.2 The “Sample ID” was entered. The sample ID was                derived from incoming specimen information.        -   7.2.3.3 Dilution parameters were set up.            -   7.2.3.3.1 When no other dilution was made besides the                1:1 mixture, the dilution factor was 2.            -   7.2.3.3.2 When a dilution was made prior to the final                1:1 mixture, the dilution factor was 2 times of the                prior dilution.            -   7.2.3.3.3 The dilution factor was updated according to                the mixture used in the dilution section of the screen.                The pencil icon was selected to bring up the dialog                screens.            -   7.2.3.3.4 The F1 Image and F2 Image sections were                verified to be identical to each other.            -   7.2.3.3.5 The “Save” button was selected after set up                was completed.

7.3 Cell Counting

-   -   7.3.1 The plastic backing from both sides of a Cellometer        counting chamber slide (SD100) was removed and placed on top of        a clean, lint-free wipe.    -   7.3.2 After the cell suspension was prepared, a small aliquot of        the sample was removed and transferred into a well of a        multiwell cell culture plate or tube.    -   7.3.3 When the sample was diluted, the dilution was performed        using cell culture medium.    -   7.3.4 20 μl of cell suspension was added into a well of the        multiwell cell culture plate or tube.    -   7.3.5 20 μl of 0.2% trypan blue or the AOPI solution was added        to the 20 μl of cell suspension and mix sample thoroughly.    -   7.3.6 20 μl of the 1:1 solution was measured and transferred it        into one side of the counting chamber.

NOTE: Touching the clear area of the slide was avoided.

-   -   7.3.7 When necessary, the sample was repeated on the other side        of the slide. 7.3.8. The chamber was inserted into the slot on        the front of the Cellometer.    -   7.3.8 For the AOPI cell counting, “Preview F1” was selected on        the main screen to preview the green fluorescent image (live        cell) image. For Trypan blue counting, “Preview Brightfield” was        selected.    -   7.3.9 The focusing wheel was used to bring image into optimal        focus. Cells had a bright center and a clearly-defined edge.    -   7.3.10 “Count” was selected to begin the counting process.    -   7.3.11 Results were displayed in a counting results pop-up box        on the computer screen that showed the results of the counting        process.

Example 4: Preparation of IL-2 Stock Solution (Cellgenix)

This example describes an exemplary preparation procedure for an IL-2stock solution.

Definitions/Abbreviations

μL: microliter or μl

BSC: Biological Safety Cabinet

BSL2: Biosafety Level 2

D-PBS: Dulbecco's Phosphate Buffered Saline

G: Gauge

GMP: Good Manufacturing Processing

HAc: Acetic Acid

HSA: Human Serum Albumin

mL: Milliliter

NA: Not applicable

PPE: Personal Protective Equipment

rhIL-2; IL-2: Recombinant human Interleukin-2

COA: Certificate of Analysis

6. Procedure

-   -   6.1 Prepared 0.2% Acetic Acid solution (HAc).    -   6.1.1 Transferred 29 mL sterile water to a 50 mL conical tube.    -   6.1.2 Added 1 mL 1 N acetic acid to the 50 mL conical tube.    -   6.1.3 Mixed well by inverting tube 2-3 times.    -   6.1.4 Sterilized the HAc solution by filtration using a        Steriflip filter.    -   6.1.5 Capped, dated and labeled the solution “Sterile 0.2%        Acetic Acid Solution”    -   6.1.6 Solution expired after 2 months. Stored at room        temperature.

6.2 Prepared 1% HSA in PBS.

-   -   6.2.1 Added 4 mL of 25% HSA stock solution to 96 mL PBS in a 150        mL sterile filter unit.    -   6.2.2 Filtered solution.    -   6.2.3 Capped, dated and labeled the solution “1% HSA in PBS.”    -   6.2.4 Solution expired after 2 months. Stored 4° C.

6.3 For each vial of rhIL-2 prepared, document.

-   -   6.4 Prepared rhIL-2 stock solution (6×10⁶ IU/mL final        concentration)        -   6.4.1 Each lot of rhIL-2 was different and required            information found in the manufacturer's Certificate of            Analysis (COA), such as:            -   6.4.1.1 Mass of rhIL-2 per vial (mg)            -   6.4.1.2 Specific activity of rhIL-2 (IU/mg)            -   6.4.1.3 Recommended 0.2% HAc reconstitution volume (mL)        -   6.4.2 Calculated the volume of 1% HSA required for rhIL-2            lot by using the equation below:

${\left( \frac{\begin{matrix}{{Vial}\mspace{14mu}{mass}\mspace{14mu}({mg}) \times} \\{{Biological}\mspace{14mu}{Activity}\mspace{14mu}\left( \frac{IU}{mg} \right)}\end{matrix}}{6 \times 10^{6}\mspace{14mu}\frac{IU}{mL}} \right) - {{HAc}\mspace{14mu}{vol}\mspace{14mu}({mL})}} = {1\%\mspace{14mu}{HSA}\mspace{14mu}{vol}\mspace{14mu}({mL})}$

-   -   -   -   6.4.2.1 For example, according to CellGenix's rhIL-2 lot                10200121 COA, the specific activity for the 1 mg vial                was 25×10⁶ lU/mg. It recommends reconstituting the                rhIL-2 in 2 mL 0.2% HAc.

${\left( \frac{1\mspace{14mu}{mg} \times 25 \times 10^{6}\mspace{14mu}\frac{IU}{mg}}{6 \times 10^{6}\mspace{14mu}\frac{IU}{mL}} \right) - {2\mspace{14mu}{mL}}} = {2.167\mspace{14mu}{mL}\mspace{14mu}{HSA}}$

-   -   -   6.4.3 Wiped rubber stopper of IL-2 vial with alcohol wipe.        -   6.4.4 Using a 16 G needle attached to a 3 mL syringe, the            recommended volume of 0.2% HAc was injected into the vial.            Care was taken to not dislodge the stopper as the needle was            withdrawn.        -   6.4.5 Inverted vial 3 times and swirled until all powder was            dissolved.        -   6.4.6 The stopper was carefully removed and set aside on an            alcohol wipe.        -   6.4.7 Added the calculated volume of 1% HSA to the vial.        -   6.4.8 Capped the vial with the rubber stopper.

    -   6.5 Storage of rhIL-2 solution        -   6.5.1 For short-term storage (<72 hrs), vials were stored at            4° C.        -   6.5.2 For long-term storage (>72 hrs), the vial was            aliquoted into smaller volumes and stored in cryovials at            −20° C. until ready to use. Freeze/thaw cycles were avoided.            Expired 6 months after date of preparation.        -   6.5.3 Rh-IL-2 labels included vendor and catalog number, lot            number, expiration date, operator initials, concentration            and volume of aliquot.

Example 5: Preparation of Media for Pre-Rep and Rep Processes

This Example describes the procedure for the preparation of tissueculture media for use in protocols involving the culture of tumorinfiltrating lymphocytes (TIL) derived from various tumor typesincluding, but not limited to, metastatic melanoma, head and necksquamous cell carcinoma, ovarian carcinoma, triple-negative breastcarcinoma, and lung adenocarcinoma. In many cases, this media was usedfor preparation of any of the TILs described in the present applicationand Examples.

Definition

μg microgram

μm micrometer

μM micromolar

AIM-V® serum-free tissue culture medium (Thermo Fisher Scientific)

BSC Biological Safety Cabinet

CM1 Complete Medium #1

CM2 Complete Medium #2

CM3 Complete Medium #3

CM4 Complete Medium #4

IU or U International units

ml milliliter

mM millimolar

NA not applicable

PPE personal protective equipment

Pre-REP pre-Rapid Expansion Process

REP Rapid Expansion Process

rhIL-2, IL-2 recombinant human Interleukin-2

RPMI1640 Roswell Park Memorial Institute medium, formulation 1640

SOP Standard Operating Procedure

TIL tumor infiltrating lymphocytes

7. Procedure

-   -   7.1 All procedures were done using sterile technique in a BSC        (Class II, Type A2).        -   7.1.1 Surface of hood was sprayed with 70% ethanol prior to            its use.        -   7.1.2 All items and reagents were sprayed with 70% ethanol            prior to placing    -   them into tissue culture hood.    -   7.2 Aliquotting of 200 mM L-glutamine        -   7.2.1 L-glutamine was supplied in larger volumes than needed            for the preparation of serum (e.g., 100 ml or 500 ml            volumes).        -   7.2.2 Thawed bottle of L-glutamine in 37° C. water bath.        -   7.2.3 Mixed L-glutamine well after thawing, as it            precipitates after thaw. Ensure that all precipitates have            returned to solution prior to aliquotting.        -   7.2.4 Placed 5-10 ml aliquots of L-glutamine into sterile 15            ml conical tubes.        -   7.2.5 Labeled tubes with concentration, vendor, lot number,            date aliquotted, and expiration date.        -   7.2.6 Tubes were stored at −20° C. and pulled as needed for            media preparation.    -   7.3 Preparation of CM1        -   7.3.1 Removed the following reagents from cold storage and            warmed them in a 37° C. water bathe:            -   7.3.1.1 RPMI1640            -   7.3.1.2 Human AB serum            -   7.3.1.3 200 mM L-glutamine        -   7.3.2 Removed the BME from 4° C. storage and place in tissue            culture hood.        -   7.3.3 Placed the gentamycin stock solution from room            temperature storage into tissue culture hood.        -   7.3.4 Prepared CM1 medium according to Table 1 below by            adding each of the ingredients into the top section of a 0.2            μm filter unit appropriate to the volume that was filtered.

TABLE 11 Preparation of CM1 Final Final Final Ingredient concentrationVolume 500 ml Volume IL RPMI1640 NA 450 ml 900 ml  Human AB serum, 50 ml100 ml heat-inactivated 10% 200 mM L-glutamine 2 mM  5 ml 10 ml  55 mMBME 55 μM  0.5 ml 1 ml 50 mg/ml gentamicin 50 μg/ml  0.5 ml 1 ml sulfate

-   -   -   7.3.5 Labeled the CM1 media bottle with its name, the            initials of the preparer, the date it was filtered/prepared,            the two week expiration date and stored at 4° C. until            needed for tissue culture. Media was aliquotted into smalled            volume bottles as required.        -   7.3.6 Any remaining RPMI1640, Human AB serum, or L-glutamine            was stored at 4° C. until next preparation of media.        -   7.3.7 Stock bottle of BME was returned to 4° C. storage.        -   7.3.8 Stock bottle of gentamicin was returned to its proper            RT storage location.        -   7.3.9 Because of the limited buffering capacity of the            medium, CM1 was discarded no more than two weeks after            preparation, or as the phenol red pH indicator showed an            extreme shift in pH (bright red to pink coloration).        -   7.3.10 On the day of use, the required amount of CM1 was            warmed in a 37° C. water bath and 6000 IU/ml IL-2 was added.        -   7.3.11 Additional supplementation—as was needed            -   7.3.11.1 CM1 was supplemented with GlutaMAX®                -   7.3.11.1.1 CM1 was prepared by substituting 2 mM                    GlutaMAX™ for 2 mM glutamine (final concentration,                    see Table 2.) When this was done, the media bottle                    was labeled adding “2 mM GlutaMAX” to prevent                    confusion with the standard formulation of CM1.            -   7.3.11.2 CM1 was supplemented with extra                antibiotic/antimycotic                -   7.3.11.2.1 Some CM1 formulations required additional                    antibiotic or antimycotic to prevent contamination                    of pre-REP TIL grown from certain tumor types.                -   7.3.11.2.2 Antibiotic/antimycotic was added to the                    final concentrations shown in Table 2 below.                -   7.3.11.2.3 When done, the media bottle was labeled                    by adding the name/s of the additional                    antibiotic/antimycotic to prevent confusion with the                    standard formulation of CM1.

TABLE 12 Additional supplementation of CM1, as was needed. SupplementStock concentration Dilution Final concentration GlutaMAXTm 200 mM 1:1002 mM Penicillin/ 10,000 U/ml 1:100 100 U/ml streptomycin penicillinpenicillin 10,000 μg/ml 100 μg/ml streptomycin streptomycin AmphotericinB 250 μg/ml 1:100 2.5 μg/ml

-   -   8.1 Preparation of CM2        -   8.1.1 Removed prepared CM1 from refrigerator or prepare            fresh CM1 as per Example above.        -   8.1.2 Removed AIM-V® from refrigerator.        -   8.1.3 Prepared the amount of CM2 needed by mixing prepared            CM1 with an equal volume of AIM-V® in a sterile media            bottle.        -   8.1.4 Added 3000 IU/ml IL-2 to CM2 medium on the day of            usage.        -   8.1.5 Made sufficient amount of CM2 with 3000 IU/ml IL-2 on            the day of usage.        -   8.1.6 Labeled the CM2 media bottle with its name, the            initials of the preparer, the date it was filtered/prepared,            the two week expiration date and stored at 4° C. until            needed for tissue culture. Media was aliquotted into smalled            volume bottles as required.        -   8.1.7 Returned any CM2 without IL-2 to the refrigerator            where it was stored for up to two weeks, or until phenol red            pH indicator showed an extreme shift in pH (bright red to            pink coloration).    -   8.2 Preparation of CM3        -   8.2.1 Prepared CM3 on the day it was required for use.        -   8.2.2 CM3 was the same as AIM-V® medium, supplemented with            3000 IU/ml IL-2 on the day of use.        -   8.2.3 Prepared an amount of CM3 sufficient to experimental            needs by adding IL-2 stock solution directly to the bottle            or bag of AIM-V. Mixed well by gentle shaking. Labeled            bottle with “3000 IU/ml IL-2” immediately after adding to            the AIM-V. When there was excess CM3, it was stored in            bottles at 4° C. labeled with the media name, the initials            of the preparer, the date the media was prepared, and its            expiration date (7 days after preparation).        -   8.2.4 Discarded media supplemented with IL-2 after 7 days            storage at 4° C.    -   8.3 Preparation of CM4        -   8.3.1 CM4 was the same as CM3, with the additional            supplement of 2 mM GlutaMAX™ (final concentration).        -   8.3.1.1 For every 1 L of CM3, added 10 ml of 200 mM            GlutaMAX™.        -   8.3.2 Prepared an amount of CM4 sufficient to experimental            needs by adding IL-2 stock solution and GlutaMAX stock            solution directly to the bottle or bag of AIM-V. Mixed well            by gentle shaking.        -   8.3.3 Labeled bottle with “3000 IL/nil IL-2 and GlutaMAX”            immediately after adding to the AIM-V.        -   8.3.4 If there was excess CM4, it was stored in bottles at            4° C. labeled with the media name, “GlutaMAX”, the initials            of the preparer, the date the media was prepared, and its            expiration date (7 days after preparation).        -   8.3.5 Discarded media supplemented with IL-2 after 7 days            storage at 4° C.

Example 6: Evaluation of Irradiated Allogeneic Feeder Cells for RapidExpansion Protocol of LN-144

This Example describes a novel abbreviated procedure for qualifyingindividual lots of gamma-irradiated peripheral mononuclear cells (PBMCs,also known as MNC) for use as allogeneic feeder cells in the exemplarymethods described herein.

Each irradiated MNC feeder lot was prepared from an individual donor.Each lot or donor was screened individually for its ability to expandTIL in the REP in the presence of purified anti-CD3 (clone OKT3)antibody and interleukin-2 (IL-2). In addition, each lot of feeder cellswas tested without the addition of TIL to verify that the received doseof gamma radiation was sufficient to render them replicationincompetent.

Definitions

-   -   AOPI—Acridine Orange/Propidum Iodide    -   BSC—Biological Safety Cabinet    -   CD3—Cluster of Differentiation 3; surface marker protein for        T-lymphocytes    -   CF—Centrifugal Force    -   CM1—Complete Medium for T1L, #1    -   CM2—Complete Medium for TIL, #    -   CMO—Contract Manufacturing Organization    -   CO₂—Carbon Dioxide    -   EtOH—Ethyl Alcohol    -   GMP—Good Manufacturing Practices    -   Gy—Gray    -   IL-2—Interleukin 2    -   IU—International Units    -   LN2—Liquid Nitrogen    -   Mini-REP—Mini-Rapid Expansion Protocol    -   ml—Milliliter    -   MNC—Mononuclear Cells    -   NA—Not Applicable    -   OKT3—MACS GMP CD3 pure (clone OKT3) antibody    -   PPE—Personal Protective Equipment    -   Pre-REP—Before Rapid Expansion Protocol    -   QS—Quantum Satis; fill to this quantity    -   REP—Rapid Expansion Protocol    -   TIL—Tumor Infiltrating Lymphocytes    -   T25—25 cm2 tissue culture flask    -   μg—Micrograms    -   μl—Microliter        Equipment, Software, Materials

Equipment

-   -   BSC (Biological Safety Cabinet)    -   Liquid Nitrogen Freezer    -   Temperature-controlled water bath    -   Centrifuge with swinging bucket rotor    -   Humidified tissue culture incubator    -   Pipet Aid    -   2-20 μl Pipettor    -   20-200 μl Pipettor    -   100-1000 μl Pipettor    -   Automated Cell Counter

Material

-   -   15 ml conical centrifuge tubes, sterile    -   50 ml conical centrifuge tubes, sterile    -   CM1    -   CM2    -   AIM V Medium CTS (Therapeutic Grade)    -   Cell Counter Staining Solution    -   IL-2    -   MACS GMP CD3 pure (clone OKT3) antibody    -   Sterile, disposable serological pipets    -   Sterile, disposable transfer pipets    -   Sterile, pipet tips    -   24-well tissue culture plate    -   T25 flasks (Greiner #690175)    -   5.3.14. Zipper storage bags        Procedure        Background

Gamma-irradiated, growth-arrested MNC feeder cells were required for REP(Step D) of TIL expansion. Membrane receptors on the feeder MNCs bind toanti-CD3 (clone OKT3) antibody and crosslink to TIL in the REP (Step D)flask, stimulating the TIL to expand. Feeder lots were prepared from theleukapheresis of whole blood taken from individual donors. Theleukapheresis product was subjected to centrifugation overFicoll-Hypaque, washed, irradiated, and cryopreserved under GMPconditions.

It was important that patients who received TIL therapy not be infusedwith viable feeder cells as this can result in Graft-Versus-Host Disease(GVHD). Feeder cells were therefore growth-arrested by dosing the cellswith gamma-irradiation, which resulted in double strand DNA breaks andthe loss of cell viability of the MNC cells upon reculture.

Evaluation Criteria and Experimental Set-Up

Feeder lots were evaluated on two criteria: 1) their ability to expandTIL in co-culture >100-fold and 2) their replication incompetency.

Feeder lots were tested in mini-REP format utilizing two primary pre-REPTIL lines grown in upright T25 tissue culture flasks. Feeder lots weretested against two distinct TIL lines, as each TIL line was unique inits ability to proliferate in response to activation in a REP. As acontrol, a lot of irradiated MNC feeder cells which was historicallybeen shown to meet the criteria of 1) and 2): (1) their ability toexpand TIL in co-culture >100-fold and (2) their replicationincompetency was run alongside the test lots.

To ensure that all lots tested in a single experiment receive equivalenttesting, sufficient stocks of the same pre-REP TIL lines were used totest all conditions and all feeder lots. For each lot of feeder cellstested, there was a total of six T25 flasks:

-   -   Pre-REP TIL line #1 (2 flasks)    -   Pre-REP TIL line #2 (2 flasks)    -   Feeder control (2 flasks)    -   NOTE: Flasks containing TIL lines #1 and #2 evaluated the        ability of the feeder lot to expand TIL. The feeder control        flasks evaluated the replication incompetence of the feeder lot.        Experimental Protocol        Day—2/3, Thaw of TIL Lines

Prepared CM2 medium as per Example 5, Pre-REP and REP Media Preparation.Warmed CM2 in 37° C. water bath. Prepared 40 ml of CM2 supplemented with3000 IU/ml IL-2. Kept warm until use. Placed 20 ml of pre-warmed CM2without IL-2 into each of two 50 ml conical tubes labeled with names ofthe TIL lines used. Removed the two designated pre-REP TIL lines fromLN2 storage and transfer the vials to the tissue culture room. RecordedTIL line identification form. Thawed vials by placing them inside asealed zipper storage bag in a 37° C. water bath until a small amount ofice remains. Sprayed or wiped thawed vials with 70% ethanol andtransferred vials to BSC. Used a sterile transfer pipet to immediatelytransfer the contents of vial into the 20 ml of CM2 in the prepared,labeled 50 ml conical tube. QS (filled to this quantity) to 40 ml usingCM2 without IL-2 to wash cells. Centrifuged at 400×CF for 5 minutes.Aspirated the supernatant and resuspended in 5 ml warm CM2 supplementedwith 3000 IU/ml IL-2. Removed small aliquot (20 μl) in duplicate forcell counting using an automated cell counter. Recorded the counts.While counting, placed the 50 ml conical tube with TIL cells into ahumidified 37° C., 5% CO₂ incubator, with the cap loosened to allow forgas exchange. Determined cell concentration and dilute TIL to 1×10⁶cells/ml in CM2 supplemented with IL-2 at 3000 IU/ml. Cultured in 2ml/well of a 24-well tissue culture plate in as many wells as needed ina humidified 37° C. incubator until Day 0 of the mini-REP. Cultured thedifferent TIL lines in separate 24-well tissue culture plates to avoidconfusion and potential cross-contamination.

Day 0, Initiate Mini-REP

Prepared enough CM2 medium for the number of feeder lots to be tested.(e.g., for testing 4 feeder lots at one time, prepare 800 ml of CM2medium). Aliquoted a portion of the CM2 prepared in Example 5 andsupplemented it with 3000 IU/ml IL-2 for the culturing of the cells.(e.g., for testing 4 feeder lots at one time, prepare 500 ml of CM2medium with 3000 IU/ml IL-2). The remainder of the CM2 with no IL-2 wasused for washing of cells as described below.

Prepared TIL

-   -   7.3.2.4. Working with each TIL line separately to prevent        cross-contamination, the 24-well plate with TIL culture was        removed from the incubator and transferred to the BSC.    -   7.3.2.5. Using a sterile transfer pipet or 100-1000 μl Pipettor        and tip, removed about 1 ml of medium from each well of TIL to        be used and placed in an unused well of the 24-well tissue        culture plate. This was used for washing wells.    -   7.3.2.6. Using a fresh sterile transfer pipet or 100-1000 μl        Pipettor and tip, mixed remaining medium with TIL in wells to        resuspend the cells and then transferred the cell suspension to        a 50 ml conical tube labeled with the TIL name and recorded the        volume.    -   7.3.2.7. Washed the wells with the reserved media and        transferred that volume to the same 50 ml conical tube.    -   7.3.2.8. Spun the cells at 400×CF to collect the cell pellet.    -   7.3.2.9. Aspirated off the media supernatant and resuspended the        cell pellet in 2-5 ml of CM2 medium containing 3000 IU/ml IL-2;        volume used was based on the number of wells harvested and the        size of the pellet—volume was sufficient to ensure a        concentration of >1.3×10⁶ cells/ml.    -   7.3.2.10. Using a serological pipet, mixed the cell suspension        thoroughly and recorded the volume.    -   7.3.2.11. Removed 200 μl for a cell count using an automated        cell counter.    -   7.3.2.12. While counting, the 50 ml conical tube with TIL cells        was placed into a humidified, 5% CO2, 37° C. incubator, with the        cap loosened to allow gas exchange.    -   7.3.2.13. Recorded the counts.    -   7.3.2.14. Removed the 50 ml conical tube containing the TIL        cells from the incubator and resuspended them cells at a        concentration of 1.3×10⁶ cells/ml in warm CM2 supplemented with        3000 IU/ml IL-2. Returned the 50 ml conical tube to the        incubator with a loosened cap.    -   7.3.2.15 When needed, the original 24-well plate was kept to        reculture any residual TIL.    -   7.3.2.16. Repeated steps 7.3.2.4-7.3.2.15 for the second TIL        line.    -   7.3.2.17. Just prior to plating the TIL into the T25 flasks for        the experiment, TIL were diluted 1:10 for a final concentration        of 1.3×10⁵ cells/ml as per step 7.3.2.35 below.        Prepare MACS GMP CD3 Pure (OKT3) Working Solution    -   7.3.2.18. Took out stock solution of OKT3 (1 mg/ml) from 4° C.        refrigerator and placed in BSC.    -   7.3.2.19. A final concentration of 30 ng/ml OKT3 was used in the        media of the mini-REP.    -   7.3.2.20. 600 ng of OKT3 were needed for 20 ml in each T25 flask        of the experiment; this is the equivalent of 60 μl of a 10 μg/ml        solution for each 20 ml, or 360 μl for all 6 flasks tested for        each feeder lot.    -   7.3.2.21. For each feeder lot tested, 400 μl of a 1:100 dilution        of 1 mg/ml OKT3 was made for a working concentration of 10 μg/ml        (e.g., for testing 4 feeder lots at one time, made 1600 μl of a        1:100 dilution of 1 mg/ml OKT3: 16 μl of 1 mg/ml OKT3+1.584 ml        of CM2 medium with 3000 IU/ml IL-2.)        Prepare T25 Flasks    -   7.3.2.22. Labeled each flask with the name of the TIL line        tested, flask replicate number, feeder lot number, date, and        initials of analyst.    -   7.3.2.23. Filled flask with the CM2 medium prior to preparing        the feeder cells.    -   7.3.2.24. Placed flasks into 37° C. humidified 5% CO2 incubator        to keep media warm while waiting to add the remaining        components.    -   7.3.2.25. Once feeder cells were prepared, the components were        added to the CM2 in each flask as shown in Table 14, Flask        Set-up, below.

TABLE 13 Flask Set-up Volume in Volume control in co- (feeder only)Component culture flasks CM2 + 3000 IU/ml IL-2 18 ml  19 ml MNC: 1.3 ×10⁷/ml in CM2 + 3000 IU 1 ml  1 ml IL-2 (final concentration 1.3 ×10⁷/flask) OKT3: 10 μg/ml in CM2 + 3000 IU IL-2 60 μl  60 μl  TIL: 1.3 ×10⁵/ml in CM2 with 3000 IU of IL-2 (final concentration 1_3 × 10⁵/flask)1 ml 0Prepared Feeder Cells

-   -   7.3.2.26. A minimum of 78×10⁶ feeder cells were needed per lot        tested for this protocol. Each 1 ml vial frozen by SDBB had        100×10⁶ viable cells upon freezing. Assuming a 50% recovery upon        thaw from LN2 storage, it was recommended to thaw at least two 1        ml vials of feeder cells per lot giving an estimated 100×10⁶        viable cells for each REP. Alternately, if supplied in 1.8 ml        vials, only one vial would provide enough feeder cells.    -   7.3.2.27. Before thawing feeder cells, pre-warmed approximately        50 ml of CM2 without IL-2 for each feeder lot to be tested.    -   7.3.2.28. Removed the designated feeder lot vials from LN2        storage, placed in zipper storage bag, and place on ice.        Transferred vials to tissue culture room.    -   7.3.2.29. Thawed vials inside closed zipper storage bag by        immersing in a 37° C. water bath.    -   7.3.2.30. Removed vials from zipper bag, spray or wipe with 70%        EtOH and transferred vials to BSC.    -   7.3.2.31. Using a transfer pipet, the contents of feeder vials        were immediately transferred into 30 ml of warm CM2 in a 50 ml        conical tube. Washed vial with a small volume of CM2 to remove        any residual cells in the vial.    -   7.3.2.32. Centrifuged at 400×CF for 5 minutes.    -   7.3.2.33. Aspirated the supernatant and resuspended in 4 ml warm        CM2 plus 3000 IU/ml IL-2.    -   7.3.2.34. Removed 200 μl for cell counting using the Automated        Cell Counter. Record the counts.    -   7.3.2.35. Resuspended cells at 1.3×10⁷ cells/ml in warm CM2 plus        3000 IU/ml IL-2.        Setup Co-Culture    -   7.3.2.36. Diluted TIL cells from 1.3×10⁶ cells/ml to 1.3×10⁵        cells/ml. Worked with each TIL line independently to prevent        cross-contamination.        -   7.3.2.36.1. Added 4.5 ml of CM2 medium to a 15 ml conical            tube.        -   7.3.2.36.2. Removed TIL cells from incubator and resuspended            well using a 10 ml serological pipet.        -   7.3.2.36.3. Removed 0.5 ml of cells from the 1.3×10⁶            cells/ml TIL suspension and add to the 4.5 ml of medium in            the 15 ml conical tube. Returned TIL stock vial to            incubator.        -   7.3.2.36.4. Mixed well.        -   7.3.2.36.5. Repeated steps 7.3.2.36.1-7.3.2.36.4 for the            second TIL line.        -   7.3.2.36.6. When testing more than one feeder lot at one            time, diluted the TIL to the lower concentration for each            feeder lot just prior to plating the TIL.    -   7.3.2.37. Transferred flasks with pre-warmed media for a single        feeder lot from the incubator to the BSC.    -   7.3.2.38. Mixed feeder cells by pipetting up and down several        times with a 1 ml pipet tip and transfer 1 ml (1.3×10⁷ cells) to        each flask for that feeder lot.    -   7.3.2.39. Added 60 μl of OKT3 working stock (10 μg/ml) to each        flask. 7.3.2.40. Returned the two control flasks to the        incubator.    -   7.3.2.41. Transferred 1 ml (1.3×10⁵) of each TIL lot to the        correspondingly labeled T25 flask.    -   7.3.2.42. Returned flasks to the incubator and incubated        upright. Did not disturb until Day 5.    -   7.3.2.43. Repeated 7.3.2.36-7.3.2.42 for all feeder lots tested.        7.3.3. Day 5, Media changed    -   7.3.3.1. Prepared CM2 with 3000 IU/ml IL-2. 10 ml is needed for        each flask    -   7.3.3.2. To prevent cross-contamination, handled the flasks for        a single feeder lot at a time. Removed flasks from the incubator        and transferred to the BSC, and care was taken not to disturb        the cell layer on the bottom of the flask.    -   7.3.3.3. Gently removed 10 ml of the media from flask and        discarded.    -   7.3.3.4. Repeated for all flasks including control flask.    -   7.3.3.5. With a 10 ml pipette, transferred 10 ml warm CM2 with        3000 IU/ml IL-2 to each flask.    -   7.3.3.6. Returned flasks to the incubator and incubate upright        until Day 7. 7.3.3.7. Repeat 7.3.3.1-7.3.3.6 for all feeder lots        tested.        7.3.4. Day 7, Harvest    -   7.3.4.1. To prevent cross-contamination, handled the flasks for        a single feeder lot at a time.    -   7.3.4.2. Removed flasks from the incubator and transferred to        the BSC, and care was taken not to disturb the cell layer on the        bottom of the flask.    -   7.3.4.3. Without disturbing the cells growing on the bottom of        the flasks, removed 10 ml of medium from each test flask and 15        ml of medium from each of the control flasks.    -   7.3.4.4. Using a 10 ml serological pipet, resuspended the cells        in the remaining medium and mixed well to break up any clumps of        cells.    -   7.3.4.5. Recorded the volumes for each flask in Day 7.    -   7.3.4.6. After thoroughly mixing cell suspension by pipetting,        removed 200 μl for cell counting.    -   7.3.4.7. Counted the TIL using the appropriate standard        operating procedure in conjunction with the automatic cell        counter equipment.    -   7.3.4.8. Recorded counts for Day 7.    -   7.3.4.9. Repeated 7.3.4.1-7.3.4.8 for all feeder lots tested.    -   7.3.4.10. Feeder control flasks were evaluated for replication        incompetence and flasks containing TIL were evaluated for fold        expansion from Day 0 according to the criteria listed in FIG. 2.        7.3.5. Day 7, Continuation of Feeder Control Flasks to Day 14    -   7.3.5.1. After completing the Day 7 counts of the feeder control        flasks, added 15 ml of fresh CM2 medium containing 3000 IU/ml        IL-2 to each of the control flasks.    -   7.3.5.2. Returned the control flasks to the incubator and        incubated in an upright position until Day 14.        7.3.6. Day 14, Extended Non-proliferation of Feeder Control        Flasks    -   7.3.6.1 To prevent cross-contamination, handled the flasks for a        single feeder lot at a time.    -   7.3.6.2 Removed flasks from the incubator and transferred to the        BSC, and care was taken not to disturb the cell layer on the        bottom of the flask.    -   7.3.6.3. Without disturbing the cells growing on the bottom of        the flasks, removed approximately 17 ml of medium from each        control flasks.    -   7.3.6.4. Using a 5 ml serological pipet, resuspended the cells        in the remaining medium and mixed well to break up any clumps of        cells.    -   7.3.6.5. Recorded the volumes for each flask.    -   7.3.6.6. After thoroughly mixing cell suspension by pipetting,        removed 200 μl for cell counting.    -   7.3.6.7. Counted the TIL using the appropriate standard        operating procedure in conjunction with the automatic cell        counter equipment.    -   7.3.6.8. Recorded counts for Day 14.    -   7.3.6.9. Repeated 7.3.4.1-7.3.4.8 for all feeder lots tested.        Expected Results and Acceptance Criteria        Expected Results

The dose of gamma irradiation was sufficient to render the feeder cellsreplication incompetent. All lots were expected to meet the evaluationcriteria and also demonstrated a reduction in the total viable number offeeder cells remaining on Day 7 of the REP culture compared to Day 0.

All feeder lots were expected to meet the evaluation criteria of100-fold expansion of TIL growth by Day 7 of the REP culture.

Day 14 counts of Feeder Control flasks were expected to continue thenon-proliferative trend seen on Day 7.

Acceptance Criteria

The following acceptance criteria had to be met for each replicate TILline tested for each lot of feeder cells.

Acceptance was Two-Fold, as Follows (Outlined in FIG. 2, AcceptanceCriteria):

Whether the dose of radiation was sufficient to render the MNC feedercells replication incompetent when cultured in the presence of 30 ng/mlOKT3 antibody and 3000 IU/ml IL-2 was evaluated.

Replication incompetence was evaluated by total viable cell count (TVC)as determined by automated cell counting on Day 7 and Day 14 of the REP.

Acceptance criteria is “No Growth,” meaning the total viable cell numberhad not increased on Day 7 and Day 14 from the initial viable cellnumber put into culture on Day 0 of the REP.

Evaluate the Ability of the Feeder Cells to Support TIL Expansion.

TIL growth was measured in terms of fold expansion of viable cells fromthe onset of culture on Day 0 of the REP to Day 7 of the REP.

On Day 7, TIL cultures achieved a minimum of 100-fold expansion, (i.e.,greater than 100 times the number of total viable TIL cells put intoculture on REP Day 0), as evaluated by automated cell counting.

MNC feeder lots that did not meet these two criteria above weretypically excluded.

Any MNC feeder lots that meet acceptance criteria but are judged to havepoor performance in regard to the ability to expand TIL relative toother previous feeder lots tested in parallel with the same pre-REP TILlines, as judged by those of skill in the art could have been excluded.See Table 15 below for acceptance criteria used.

TABLE 14 Acceptance Criteria Test Acceptance criteria Irradiation ofMNC/ No growth observed at 7 and 14 days Replication Incompetence TILexpansion At least a 100-fold expansion of each TIL (minimum of 1.3 ×10⁷ viable cells)

Whether the dose of radiation was sufficient to render the MNC feedercells replication incompetent when cultured in the presence of 30 ng/mlOKT3 antibody and 3000 IU/ml IL-2 was evaluated.

-   -   10.2.2.1.1 Replication incompetence was evaluated by total        viable cell count (TVC) as determined by automated cell counting        on Day 7 and Day 14 of the REP.    -   10.2.2.1.2 Acceptance criteria was “No Growth,” meaning the        total viable cell number was not increased on Day 7 and Day 14        from the initial viable cell number put into culture on Day 0 of        the REP.    -   10.2.2.2 The ability of the feeder cells to support TIL        expansion was evaluated.    -   10.2.2.2.1 TIL growth was measured in terms of fold expansion of        viable cells from the onset of culture on Day 0 of the REP to        Day 7 of the REP.    -   10.2.2.2.1 On Day 7, TIL cultures achieved a minimum of 100-fold        expansion, (i.e., greater than 100 times the number of total        viable TIL cells put into culture on REP Day 0), as evaluated by        automated cell counting.    -   10.2.2.3 When a lot failed to meet the two criteria above, the        lot was retested according to the contingency plan outlined in        Section 10.3 below.    -   10.2.2.4 Following retesting of a failed lot, any MNC feeder lot        that did not meet the two acceptance criteria in both the        original evaluation and the contingency testing was excluded.    -   10.2.2.5 Any MNC feeder lots that met acceptance criteria but        were judged to have poor performance in regard to the ability to        expand TIL relative to other previous feeder lots tested in        parallel with the same pre-REP TIL lines were excluded as        appropriate.

Contingency Testing of MNC Feeder Lots that do not meet acceptancecriteria

-   -   10.3.1 In the event that an MNC feeder lot met either of the        acceptance criteria outlined in Section 10.2 above, the        following steps were taken to retest the lot to rule out simple        experimenter error as its cause.    -   10.3.2 If there were two or more remaining satellite testing        vials of the lot, then the lot could be retested. If there were        one or no remaining satellite testing vials of the lot, then the        lot was failed according to the acceptance criteria listed in        Section 10.2 above.    -   10.3.3 Two trained personnel, include the original person who        evaluated the lot in question, had to both test the lot at the        same time.    -   10.3.4 Repeating Section 7.2-7.3 was done to re-evaluate the lot        in question.    -   10.3.5 Each person would test the lot in question as well as a        control lot (as defined in Section 7.2.4 above).    -   10.3.6 In order to be qualified, the lot in question and the        control lot had to achieve the acceptance criteria of Section        10.2 for both of the personnel doing the contingency testing.    -   10.3.7 Upon meeting these criteria, the lot could then be        released for CMO use as outlined in Section 10.2 above.

Example 7: Procedure for Qualifying Individual Lots of Gamma-IrradiatedPeripheral Blood Mononuclear Cells

This Example describes a novel abbreviated procedure for qualifyingindividual lots of gamma-irradiated peripheral blood mononuclear cells(PBMC) for use as allogeneic feeder cells in the exemplary methodsdescribed herein. This example provides a protocol for the evaluation ofirradiated PBMC cell lots for use in the production of clinical lots ofTIL. Each irradiated PBMC lot was prepared from an individual donor.Over the course of more than 100 qualification protocols, it has beenshown that, in all cases, irradiated PBMC lots from SDBB (San DiegoBlood Bank) can expand TILs >100-fold on Day 7 of a REP. This modifiedqualification protocol is intended to apply to irradiated donor PBMClots from SDBB which must still be tested to verify that the receiveddose of gamma radiation was sufficient to render them replicationincompetent. Once demonstrated that they maintain replicationincompetence over the course of 14 days, donor PBMC lots were considered“qualified” for usage to produce clinical lots of TIL.

Key Terms and Definitions

μg—Microgram

μl—Microliter

AIM-V—commercially available cell culture medium Biological SafetyCabinet

BSC—Cluster of Differentiation

CD—Complete Medium for TIL #2

CM2—CM2 supplemented with 3000 IU/ml IL-2

CM2IL2—Contract Manufacturing Organization

CO₂—Carbon Dioxide

EtOH—Ethanol

GMP—Good Manufacturing Practices

Gy—Gray

IL—Interleukin

IU—International Units

LN2—Liquid Nitrogen

MI—Milliliter

NA—Not Applicable

OKT3—anti-CD3 monoclonal antibody designation

P20—2-20 μl pipettor

P200—20-200 μl pipettor

PBMC—peripheral blood mononuclear cells

P1000—100-1000 μl pipettor

PPE—Personal Protective Equipment

REP—Rapid Expansion Protocol

SDBB—San Diego Blood Bank

TIL—Tumor Infiltrating Lymphocytes

T25—25 cm2 tissue culture flask

×g—“times gravity”—measure of relative centrifugal force

Specimens included Irradiated donor PBMC (SDBB).

Procedure

Background

-   -   7.1.1 Gamma-irradiated, growth-arrested PBMC were required for        current standard REP of TIL. Membrane receptors on the PBMCs        bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in        culture, stimulating the TIL to expand. PBMC lots were prepared        from the leukapheresis of whole blood taken from individual        donors. The leukapheresis product was subjected to        centrifugation over Ficoll-Hypaque, washed, irradiated, and        cryopreserved under GMP conditions.        -   It is important that patients who receive TIL therapy not be            infused with viable PBMCs as this can result in            Graft-Versus-Host Disease (GVHD). Donor PBMCs were therefore            growth-arrested by dosing the cells with gamma-irradiation,            resulting in double strand DNA breaks and the loss of cell            viability of the PBMCs upon reculture.            Evaluation Criteria    -   7.2.1 Evaluation criterion for irradiated PBMC lots was their        replication incompetency.        Experimental Set-Up    -   7.3.1 Feeder lots were tested in mini-REP format as if they were        to be co-cultured with TIL, using upright T25 tissue culture        flasks.        -   7.3.1.1 Control lot: One lot of irradiated PBMCs, which had            historically been shown to meet the criterion of 7.2.1, was            run alongside the experimental lots as a control.    -   7.3.2 For each lot of irradiated donor PBMC tested, duplicate        flasks were run.        Experimental Protocol

All tissue culture work in this protocol was done using steriletechnique in a BSC.

Day 0

-   -   7.4.1 Prepared ˜90 ml of CM2 medium for each lot of donor PBMC        to be tested. Kept CM2 warm in 37° C. water bath.    -   7.4.2 Thawed an aliquot of 6×10⁶ IU/ml IL-2.    -   7.4.3 Returned the CM2 medium to the BSC, wiping with 70% EtOH        prior to placing in hood. For each lot of PBMC tested, about 60        ml of CM2 was removed to a separate sterile bottle. Added IL-2        from the thawed 6×10⁶ IU/ml stock solution to this medium for a        final concentration of 3000 IU/ml. Labeled this bottle as        “CM2/IL2” (or similar) to distinguish it from the unsupplemented        CM2.    -   7.4.4 Labeled two T25 flasks for each lot of PBMC to be tested.        Minimal label included:        -   7.4.4.1 Lot number        -   7.4.4.2 Flask number (1 or 2)        -   7.4.4.3 Date of initiation of culture (Day 0)            Prepared OKT3    -   7.4.5 Took out the stock solution of anti-CD3 (OKT3) from the        4° C. refrigerator and placed in the BSC.    -   7.4.6 A final concentration of 30 ng/ml OKT3 was used in the        media of the mini-REP.    -   7.4.7 Prepared a 10 μg/ml working solution of anti-CD3 (OKT3)        from the 1 mg/ml stock solution. Placed in refrigerator until        needed.        -   7.4.7.1 For each PBMC lot tested, prepared 150 μl of a 1:100            dilution of the anti-CD3 (OKT3) stock.            -   E.g., for testing 4 PBMC lots at one time, prepared 600                μl of 10 μg/ml anti-CD3 (OKT3) by adding 6 μl of the 1                mg/ml stock solution to 594 μl of CM2 supplemented with                3000 IU/ml IL-2.                Prepared Flasks    -   7.4.8 Added 19 ml per flask of CM2/IL-2 to the labeled T25        flasks and place flasks into 37° C., humidified, 5% CO₂        incubator while preparing cells.        Prepared Irradiated PBMC    -   7.4.9 Worked with each donor PBMC lot individually to avoid the        potential cross-contamination of the lots.    -   7.4.10 Retrieved vials of PBMC lots to be tested from LN2        storage. These were placed at −80° C. or kept on dry ice prior        to thawing.    -   7.4.11 Placed 30 ml of CM2 (without IL-2 supplement) into 50 ml        conical tubes for each lot to be thawed. Labeled each tube with        the different lot numbers of the PBMC to be thawed. Capped tubes        tightly and place in 37° C. water bath prior to use. As needed,        returned 50 ml conical tubes to the BSC, wiping with 70% EtOH        prior to placing in the hood.    -   7.4.12 Removed a vial PBMC from cold storage and place in a        floating tube rack in a 37° C. water bath to thaw. Allowed thaw        to proceed until a small amount of ice remains in the vial.    -   7.4.13 Sprayed or wiped thawed vial with 70% EtOH and transfer        to BSC.    -   7.4.14 Using a sterile transfer pipet, the contents of the vial        were immediately transferred into the 30 ml of CM2 in the 50 ml        conical tube. Removed about 1 ml of medium from the tube to        rinse the vial; returned rinse to the 50 ml conical tube. Capped        tightly and swirl gently to wash cells.    -   7.4.15 Centrifuged at 400×g for 5 min at room temperature.    -   7.4.16 Aspirated the supernatant and resuspended the cell pellet        in 1 ml of warm CM2/IL-2 using a 1000 μl pipet tip.        Alternatively, prior to adding medium, resuspended cell pellet        by dragging capped tube along an empty tube rack. After        resuspending the cell pellet, bring volume to 4 ml using        CM2/IL-2 medium. Recorded volume.    -   7.4.17 Removed a small aliquot (e.g., 100 μl) for cell counting        using an automated cell counter.        -   7.4.17.1 Performed counts in duplicate according to the            particular automated cell counter SOP. It was often            necessary to perform a dilution of the PBMC prior to            performing the cell counts. A recommended starting dilution            was 1:10, but this could vary depending on the type of cell            counter used.        -   7.4.17.2 Recorded the counts.    -   7.4.18 Adjusted concentration of PBMC to 1.3×10⁷ cells/ml as per        step 7.4.15.2 using CM2/IL-2 medium. Mixed well by gentle        swirling or by gently aspirating up-and-down using a serological        pipet.        Set Up Culture Flasks    -   7.4.19 Returned two labeled T25 flasks to the BSC from the        tissue culture incubator.    -   7.4.20 Returned the 10 μg/ml vial of anti-CD3/OKT3 to the BSC.    -   7.4.21 Added 1 ml of the 1.3×10⁷ PBMC cell suspension to each        flask.    -   7.4.22 Added 60 μl of the 10 μg/ml anti-CD3/OKT3 to each flask.    -   7.4.23 Returned capped flasks to the tissue culture incubators        for 14 days of growth without disturbance.    -   7.4.24 The anti-CD3/OKT3 vial was placed back into the        refrigerator until needed for the next lot.    -   7.4.25 Repeated steps 7.4.9-7.4.24 for each lot of PBMC to be        evaluated.        Day 14, Measurement of Non-Proliferation of PBMC    -   7.4.26 Working with each lot independently, carefully returned        the duplicate T25 flasks to the BSC.    -   7.4.27 For each flask, using a fresh 10 ml serological pipet,        removed ˜17 ml from each of the flasks, then carefully pulled up        the remaining media to measure the volume remaining in the        flasks. Recorded volume.    -   7.4.28 Mixed sample well by pipetting up and down using the same        serological pipet.    -   7.4.29 Removed a 200 μl sample from each flask for counting.    -   7.4.30 Counted cells using an automated cell counter.    -   7.4.31 Repeated steps 7.4.26-7.4.31 for each lot of PBMC being        evaluated.        Results and Acceptance Criterion        Results    -   10.1.1 The dose of gamma irradiation was sufficient to render        the feeder cells replication incompetent. All lots were expected        to meet the evaluation criterion and demonstrated a reduction in        the total viable number of feeder cells remaining on Day 14 of        the REP culture compared to Day 0.        Acceptance Criterion 10.2.1 The following acceptance criterion        was met for each irradiated donor PBMC lot tested:    -   10.2.2 “No growth”—meaning that the total number of viable cells        on Day 14 was less than the initial viable cell number put into        culture on Day 0 of the REP.    -   10.2.3 Should a lot fail to meet the criterion above, the lot        was retested per the Contingency Testing Procedure outlined in        the section 10.4.    -   10.2.4 Following retesting of a failed lot, any MNC feeder lot        that did not meet the acceptance criterion in both the original        evaluation and the contingency testing was excluded.        Contingency Testing of PBMC Lots which Did not Meet Acceptance        Criterion.    -   10.4.1 In the event than an irradiated donor PBMC lot did not        meet the acceptance criterion above, the following steps were        taken to retest the lot to rule out simple experimenter error as        the cause of its failure.    -   10.4.2 If there were two or more remaining satellite vials of        the lot, then the lot was retested. If there were one or no        remaining satellite vials of the lot, then the lot was failed        according to the acceptance criterion of section 10.2 above.    -   10.4.3 Whenever possible, two trained personnel (preferably        including the original person who evaluated the lot in question)        did the testing of the two separate vials independently. This        was the preferred method of contingency testing. Aside from the        separate vials of PBMC, the same reagents can be used by both        personnel.        -   10.4.3.1. If two personnel were not available, one person            did the testing of the two PBMC vials for the failed lot,            working with each vial independently.    -   10.4.4 Repeating of section 7.4 “Experimental Protocol” was done        to re-evaluated the lot in question.    -   10.4.5 In addition to the lot in question, a control lot was        tested by each person carrying out the contingency testing.        -   10.4.5.1 If two personnel perform contingency testing, both            personnel tested the control lot independently.        -   10.4.5.2 If only one person was available to perform            contingency testing, it was not necessary for the control            lot to be run in duplicate.        -   10.4.5.3 To be qualified, a PBMC lot going through            contingency testing must have had both the control lot and            both replicates of the lot in question achieve the            acceptance criterion of Section 10.2 to pass.        -   10.4.5.4 Upon meeting this criterion, the lot was then be            released for CMO usage as outlined in section 10.2.

Example 8: Comparison of Pre- and Post-Cryopreserved TILS

Antibody cocktails for the samples and the FMO controls were made beforestarting the sample preparation and staining procedure. The cocktailswere stored at 4° C. in the dark for up to 60 days. See CocktailPreparation section below.

TABLE 15 Staining Procedure: Step Description 1 Removed Aqua dye aliquotfrom the freezer.ptdark. 2 Added 3 mL 1xPBS to each sample tube 3 Spuntubes at 300 g for 5 minutes. 4 Prepared Aqua Live/Dead stain. Dilute1:200 in PBS. 25 μL per sample and FMO control tube is needed. 1:200 =    μL Aqua +     mL PBS 5 Aspirated or decanted supernatant from step3. 6 Added 25 μL of Aqua L/D to each sample tube. Resuspended cells bydragging along rack. Incubated 15 min., dark, room temperature. 7Without washing, added 50 μL of appropriate Ab cocktail to each tube. 8Incubated tubes for 15 minutes at room temperature. 9 Added 3 mLs ofFACS Wash buffer 10 Spun at 330 g for 5 min at 4° C. 11 Resuspendedtubes by dragging along an empty tube rack. 12 Added 100 μL 1% PFA/PBSsolution at 4° C. 13 Stored samples at 4° C. in dark for up to 72 hours.14 Ran samples on Flow Cytometer

TABLE 16 Differentiation Panel 1 (DF1): Catalog Target Format CloneSupplier Number Titre TCRab PE/Cy7 IP26 BioLegend 306720 3 CD57*PerCP-Cy5.5 HNK-1 BioLegend 359622 2 CD28* PE CD28.2 BioLegend 302908 2CD4 FITC OKT4 eBioscience 11-0048-42 2 CD27* APC-H7 M-T271 BD 560222 3Biosciences CD56 APC N901 Beckman IM2474U 3 Coulter CD8a PB RPA-T8BioLegend 301033 2 FACS Buffer 33

TABLE 17 Differentiation Panel 2 (DF2): Catalog Target Format CloneSupplier Number Titre CD45RA* PE-Cy7 HI100 BD 560675 1 Biosciences CD8aPerCP/Cy5.5 RPA-T8 BioLegend 301032 2 CCR7* PE 150503 BD 560765 5Biosciences CD4 FITC OKT4 eBioscience 11-0048-42 2 CD3 APC/Cy7 HIT3aBioLegend 300318 2 CD38* APC HB-7 BioLegend 356606 1 HLA-DR PB L243BioLegend 307633 2 FACS Buffer 35 *Denotes FMO (Fluorescence Minus One)control should be made.

TABLE 18 T cell Activation Panel 1 (Tact1) Catalog Target Format CloneSupplier Number Titre CD137* PE/Cy7 4B4-1 BioLegend 309818 2 CD8aPerCP/Cy5.5 RPA-T8 BioLegend 301032 2 Lag3* PE 3DS223H eBioscience12-2239-42 5 CD4 FITC OKT4 BioLegend 317408 2 CD3 APC/Cy7 HIT3aBioLegend 300318 1 PD1* APC EH12.2H7 BioLegend 329908 2 Tim-3* BV421F38-2E2 BioLegend 345008 2 FACS Buffer 34

TABLE 19 T cell Activation Panel 2 (Tact2) Catalog Target Format CloneSupplier Number Titre CD69* PE-Cy7 FN50 BD 557745 3 Biosciences CD8aPerCP/Cy5.5 RPA-T8 BioLegend 301032 2 TIGIT* PE MBSA43 eBioscience12-9500-42 3 CD4 FITC OKT4 BioLegend 317408 2 CD3 APC/Cy7 HIT3aBioLegend 300318 2 KLRG1* Ax647 SA231A2 BioLegend 367704 1 CD154* BV421TRAP1 BD 563886 3 Biosciences FACS Buffer 34 *Denotes FMO (FluorescenceMinus One) control should be made.Compensation Controls

1. Added one drop of BD Comp beads to 11 tubes.

2. Labeled tubes 1 through 7 with the chromophores from DF1

3. Labeled tubes 8 through ten with APCy7, BV421, and Ax647.

4. Tube 11 was for unlabeled beads.

5. Added 5 μL of Antibody to each tube.

6. Incubated 10 to 30 minutes in dark, room temperature.

7. Washed with 3 mLs FACS Buffer

8. Resuspended with 500 uL 1% PFA.

9. Added one drop of BD Comp negative bead to each tube.

10. Stored at 4° C. in dark. Could be used for one week.

Aqua Control:

1. Added one drop of Arc positive control to tube labelled Aqua.

2. Added 34 of thawed aqua solution to tube.

3. Repeated steps 6-10 as above. Except used the negative Arc bead forstep 9.

TABLE 20 Setup. Tube Target Format Titre 1 TCRab PE/Cy7 5 2 CD57PerCP-Cy5.5 5 3 CD28 PE 5 4 CD4 FITC 5 5 CD27 APC-H7 5 6 CD56 APC 5 7CD8a PB 5 8 CD3 APC/Cy7 5 9 Tim-3 BV421 5 10 KLRG1 Ax647 5 11 Unlabeledn/a n/a

Example 9: Remarkably Stable Tumor-Infiltrating Lymphocytes (TIL) forInfusion Phenotype Following Cryopreservation

Abstract Background:

This Example discusses the development of cancer immunotherapies basedon tumor-infiltrating lymphocytes (TIL) with the ultimate goal ofdeveloping therapeutic populations of TILs. Cryopreservation of TILsallows the final cell product to be shipped in a safe manner with fewertemporal constraints (Axelsson S, Faresjo M, Hedman M, Ludvigsson J,Casas R: Cryopreserved peripheral blood mononuclear cells are suitablefor the assessment of immunological markers in type 1 diabetic children.Cryobiology 2008, 57:201-8.)

Here, fresh versus frozen/thawed TIL samples were evaluated for theexpression of individual phenotypic markers to assess whether phenotypicchanges occur with cryopreserved TILs. (See, for example, Sadeghi A,Ullenhag G, Wagenius G, Tötterman T H, Eriksson F: Rapid expansion of Tcells: Effects of culture and cryopreservation and importance ofshort-term cell recovery. Acta Oncol. 2013, 52:978-86.)

Results:

No significant differences in CD4, CD8, NK, TCRαβ expression, or memorymarkers comparing fresh versus thawed TIL were observed. The activationstatus of TIL as defined by HLA-DR, CD38, and CD69 expression wasmaintained while regulatory molecules LAG-3 and TIM-3 demonstrated aslight decrease in expression. In addition, the viability of both thefresh and thawed product was greater than 86%.

Methods:

PreREP TILs were obtained by culturing melanoma tumor fragments in IL-2(6000 IU/ml).

Rapid Expansion Protocol (REP) cells were initiated using irradiatedallogeneic PBMC feeder cells with OKT3 and IL-2 in a GREX-100 flask for11-14 days.

Cultured cells were cryopreserved in 5% DMSO.

Flow cytometric evaluation of fresh and thawed TIL following rest for 1to 2 hours in IL-2 was performed using four panels consisting oflineage, differentiation, activation, and regulatory markers.

Conclusion:

Cryopreservation did not affect the measured phenotypic characteristicsof TIL, with the exception of modest changes in some regulatorymolecules. We are investigating the possibility of using cryopreservedTIL in a clinical setting.

Example 10: Memory Cell Subsets in Fresh Versus REREP TIL Populations

In previous experiments, no central memory subset was seen with freshTIL populations (see, FIG. 8). However, after the ReREP nearly 60%central memory cells, as provided in Table 22 below.

Based on the raw numbers, the rested cells had a slightly higher CD4population than the not rested. Overall the CD8 percentage was high asexpected. It's roughly a 60/40 split for CM (central memory—Q3)/EM(effector memory—Q4) among the CD8s. The CD8+CD28+ expression looksinteresting. The rested cells have a higher amount. See also, FIGS. 9and 10A-10B. See, also FIG. 15.

Example 11: Administration of Autologous Tumor Infiltrating Lymphocytes(TILS) in Melanoma Patients

Administration of autologous tumor infiltrating lymphocytes (TILs) inmelanoma patients has shown an overall response of 55% at NCI, 38% atMoffitt Cancer Center, 48% at MD Anderson Cancer Center, and 40% inSheba at the Ella Cancer Institute, Israel. The durable responsesobserved in melanoma patients using ACT may permit broader applicationto other solid tumors. As shown herein, the feasibility of growing TILsand developing TIL therapies for other solid tumors is demonstrated. Theexample provides data showing “Successful expansion and characterizationof tumor infiltrating lymphocytes (TILs) from non-melanoma tumors.”,see, FIGS. 12-14.

Phenotypic characterization of TILs from bladder, cervical, and lungcancer were greater than 60-70% CD8+ T-cells whereas TILs from head anddemonstrated variable distribution of CD8+ and CD4+ T-cells. TILspropagated from TNBC were greater than 80% CD4+ T-cells. Regardless ofthe tumors, most cultures had less than 20% CD56+NK cells.

TILs were prepared by:

-   -   a. Washing an obtained tumor in HBSS;    -   b. Dicing the tumor into fragments (e.g., 2-3 mm3 fragments);    -   c. Placing the tumor fragments in G-REX 10 cell culture flasks        with medium containing serum and IL-2;    -   d. Exchanging media on day 7 and every 4-5 days from day 11        until day 21; and    -   e. Assessing cell count, viability, and phenotyping followed by        cryopreservation for future purposes including, but not limited        to, future delivery to patients for the treatment of tumors, as        described herein.

As demonstrated herein, TILs were grown from lung, bladder, head andneck, cervical, and TNBC patient tumors.

Moreover, as demonstrated herein, lung, bladder, and cervical tumorsshowed greater proportion of CD8+ TILs. Head and neck and TNBC tumorswere mostly CD4+ TILs. In addition, further characterization of CD4+ andCD8+ TILs demonstrated effector memory phenotypic cells that were alsoCD27+ and CD28+.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

The invention claimed is:
 1. A method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs, the methodcomprising: (a) performing a first expansion by (i) thawingcryopreserved dissociated tumor materials comprising a first populationof TILs obtained from a tumor that was resected from a subject, and (ii)culturing the first population of TILs in a cell culture mediumcomprising IL-2 to produce a second population of TILs; (b) performing asecond expansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, OKT-3, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the thirdpopulation of TILs is a therapeutic population of TILs, and wherein thesecond expansion is performed for about 14 to 22 days in order to obtainthe third population of TILs; (c) harvesting the third population ofTILs obtained from step (b); (d) transferring the harvested thirdpopulation of TILs from step (c) into an infusion bag; and (e)cryopreserving the infusion bag comprising the harvested TIL populationfrom step (d) using a cryopreservation process.
 2. The method accordingto claim 1, wherein the dissociated tumor materials comprise a tumordigest.
 3. The method according to claim 1, wherein obtaining thedissociated tumor materials comprises fragmenting the tumor resectedfrom the subject into one or more tumor fragments.
 4. The methodaccording to claim 1, wherein obtaining the dissociated tumor materialscomprises mechanically disrupting the tumor resected from the subject.5. The method according to claim 1, wherein obtaining the dissociatedtumor materials comprises enzymatically disrupting the tumor resectedfrom the subject.
 6. The method according to claim 1, wherein the cellculture medium is CTS Optimizer.
 7. The method according to claim 1,wherein the third population of TILs harvested in step (c) comprisessufficient TILs for use in administering a therapeutically effectivedosage to a subject.
 8. The method according to claim 7, wherein thetherapeutically effective dosage of TILs comprises from about 1×10⁹ toabout 9×10¹⁰ TILs.
 9. The method according to claim 1, wherein the APCscomprise peripheral blood mononuclear cells (PBMCs).
 10. The methodaccording to claim 1, wherein the PBMCs are supplemented at a ratio ofabout 1:25 TIL:PBMCs.
 11. The method according to claim 1, wherein step(a) further comprises adding the thawed dissociated tumor materials intoa closed system prior to culturing the first population of TILs.
 12. Themethod according to claim 11, wherein the transition from step (a) tostep (b), the transition from step (b) to step (c), or the transitionfrom step (c) to step (d) occurs without opening the system.
 13. Themethod according to claim 1, wherein step (a) further comprises addingthe thawed dissociated tumor materials into a closed system prior toculturing the first population of TILs, wherein the transition from step(a) to step (b) occurs without opening the system, wherein thetransition from step (b) to step (c) occurs without opening the system,and wherein the transition from step (c) to step (d) occurs withoutopening the system.
 14. The method according to claim 1, wherein thesubject has a cancer that is selected from the group consisting ofmelanoma (including metastatic melanoma), ovarian cancer, cervicalcancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer,breast cancer, cancer caused by human papilloma virus, head and neckcancer (including head and neck squamous cell carcinoma (HNSCC)), renalcancer, and renal cell carcinoma.
 15. The method according to claim 1,wherein the first expansion is performed within from about 11 to 21days.
 16. The method according to claim 1, wherein the first expansionis performed within about 11 days, 12 days, 13 days, 14 days, 15 days,16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
 17. The methodaccording to claim 1, wherein the first expansion is performed withinabout 11 days, 12 days, 13 days or 14 days.
 18. The method according toclaim 1, wherein the second expansion is performed within about 14 days,15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or 22days.
 19. The method according to claim 1, wherein the second expansionis performed within about 14 days, 15 days, 16 days or 17 days.
 20. Themethod according to claim 1, wherein the second expansion is performedwithin about 14 days or 15 days.
 21. The method according to claim 1,wherein the second expansion is performed within about 14 days.
 22. Themethod according to claim 1, wherein steps (a) through (e) are performedwithin about 24 days.
 23. The method according to claim 1, wherein thecryopreserved dissociated tumor materials were obtained bycryopreserving dissociated tumor materials in a cryopreservation media.24. The method according to claim 23, wherein the cryopreservation mediacomprises DMSO.
 25. The method according to claim 23, wherein thecryopreservation media comprises 5% DMSO.